Friday, 30 July 2021

Lupine Publishers | Microbial Source Tracking Markers for Detection of Faecal Contamination in Environmental Waters

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Mini Review

Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of faecal pollution sources in environmental waters. They rely on the association of certain fecal microorganisms with a particular host, that ranges from human [1] to agricultural animals [2,3] to pets [4] and wild animals such as gulls [5]. MST is used to appraise recreational water quality and to correlate with human health risk.

Many MST publications fixate mostly on human source contamination, as this has been an issue of concern for managers and regulators. Human sewage pollution is among the greatest concerns especially in India [7,8] for human health due to [1] the known risk of exposure to human waste and [2] the public and regulatory will to reduce sewage pollution. However, methods to identify animal sources are receiving increasing attention as our understanding of zoonotic disease potential improves.

Numerous epidemiological studies of waterborne illness in countries like India indicate that the common aetiological agents are bacteria, viruses and parasitic protozoa and the coliform presence is an important parameter for determining water pollution levels. But the presence of coliforms of soil and litter origin had raised doubt about their reliability as an indicator of the pathogen [6]. Also, the method used for coliform estimation is not precise. Hence CPCB believes that BOD is the single most reliable parameter. Based on this approach the CPCB estimated that 67% of river stretches in India is relatively clean with BOD less than 3 mg/litre. Also, poor correlations have been reported between waterborne human viruses or protozoa and thermotolerant coliforms [6,9,10]. In contrast to MST markers, faecal coliforms provided a poor metric to assess risks of exposure to faecal contamination of human origin in the rural setting in India [9]. Such a situation is critical to understand, as evident from recent drinking water outbreaks where coliform standards were met [11] and in India most of the outbreaks go unnoticed. FIB concentrations have not been well correlated with pathogens in many studies [10].

Furthermore, many epidemiology studies have failed to find a correlation between human health outcomes and FIB levels, particularly when the pollution is not from a known point source such as a wastewater treatment plant (WWTP) [14,15]. Nonetheless, water regulatory agencies have yet to come to terms with the inherent problems resulting from reliance on faecal indicator bacteria as currently determined. Fortunately, new index organisms for some pathogens look promising like C. perfringens, phages and viruses like Adenoviruses. As these index organisms are relatively untested worldwide, extensive trials are necessary before their general acceptance in microbial risk assessment. It should be noted that useful index organisms in one system are not necessarily of value in a different environment.

Use of multiple tests increases the surety of presence or absence of water pollution. In particular, it has been reported that at least two parameters are needed to accurately differentiate between two distinct faecal pollution sources: one specific indicator that identifies the source and another, a universal indicator that provides information on the faecal load [12]. In consequence, the indicators should be carefully selected based on appropriate statistical analyses. Persistence studies are needed to provide complementary information, addressing the effects of environmental aspects like temperature, solar radiation, salinity, pH, chemical pollutants, water filtration, turbidity, starvation, predation and presence of heavy metals, among others.

Other relevant issues are the use of methods based either on genomic targets or in the quantification of new microbial tracers through library-independent methods, the combination of methods, and comparative and integrated studies between research groups with standardised procedures to avoid differences in implementation. Also, in order to increase the validity of MST methods, it is necessary to consider temporal variability when designing the sampling scheme of the source material and constructing source libraries and increase the specificity and field testing of DNA-based markers. The number and range of potential host sources included in MST studies must be deliberately chosen to suit the water body and particular questions associated with it [13].

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Thursday, 29 July 2021

Lupine Publishers | Adolescent Girls’ Knowledge and Attitude About Mental Health Issues: A Questionnaire

 Lupine Publishers | Journal of Psychology and Behavioral Sciences


Background: The aims of this current study were to prepare and validate a Bengali questionnaire to assess knowledge and attitude of adolescents about the various mental health issues related to them, and to conduct a pilot study with this questionnaire.

Method:It was a cross sectional study conducted on adolescent school girls (N=107, 12 to 18years age) from rural background of West Bengal. A 13 item Bengali questionnaire was constructed, validated and administered on the subjects. Statistical analysis was done using SPSS (16th version).

Results: Most of the items had good test re-test reliability. Mean age of the population was 13.09 years. Three knowledgebased questions had more correct responses (50.5%, 52.3%, 50.5%) and one had 72% incorrect responses. Majority of the subjects recognised problem behaviours, expressed a helping attitude, tended to seek help from parents, and thought sharing worries with others and spending time with friends could make them happy.

Conclusions: This questionnaire appears reliable to assess knowledge and attitude of adolescent girls. There is need to inform adolescents about various mental health issues. Attitude to help and a sense of cohesion was conspicuous in this population. A larger and more inclusive study needed to generalize the findings.

Keywords: Adolescents; Awareness; Reliability


Adolescence and its crisis have been object of amazement and awe since ages. With the recent rise of the instances of problem behaviour like self-harm, criminal acts and various risk taking acts by adolescents, especially in our country, it has become necessary to look into the problem through an adolescents’-lens, to know about how much aware they are about the phenomenon of transformation from a child to an adult, about the various changes, challenges and problems typical of this period [1]. Part of this transition is a plethora of changes pertaining to the manner they view the things around them, about others, about themselves. Often these changes become too much for them to cope up with, resulting in various mental health issues like adjustment problems, depression, and self-harm behaviors [2]. At times problems such as pervasive unhappiness and depression, quarrelling, using abusive language and delinquent behaviour in school students, especially at secondary level, have been found to be posing serious threat to proper academic and personality development of the students [3]. Owing to a number of factors like, the different developmental trajectories for boys and girls, explained by the negative experience of intensification of stereotypical gender roles and differences in pubertal development resulting in earlier maturity of girls leading to apparent differences in processes of family influence, girls during adolescence have a typical pattern of disclosing their inner world and spending time alone or with peers and parents which is distinct from the boys in adolescence [4,5]. These, along with cultural practices, often result in problems like excessive anxiety, depression, feelings of low self-esteem, avoidant behavior and excessive dependence on others, more in adolescent girls [6].

To face the challenges of this critical period, to cope up with the crises and to deal effectively with others with those problems one need to be aware about those issues [7]. Inspite of the fact that, gathering information about their awareness of mental wellbeing and their attitude towards mental health problems forms a critical step for behavioral scientists to intervene into those situations, Little attention has been focused on promoting mental health awareness, especially in less economically developed nations where the burden is great [8,9]. Studies have suggested that direct targeting of children themselves is ideal for increasing awareness [7,10,11]. As part of ‘Adolescence Health Training Program’, a joint venture of Sarva Shiksha Mission (Dept. of School Education, Govt. of West Bengal) and Burdwan Science Centre (National Council of Science Museums, Ministry of Culture, Govt. of India), adolescent girls from different schools (class 6 to 10) were brought, in batches, in a convention centre of Burdwan (a town in West Bengal) to be addressed about the different problems of adolescence. They attended lectures by Psychiatrists and Gynecologists on different aspects of adolescents’ problems. To assess their awareness about mental health a questionnaire was needed. After searching literature, several questionnaires were found that assesses the problems and problem behaviors of adolescents, though not much was found that assesses the knowledge and attitude of adolescents about the mental health issues that are so particular of their age. This dearth seemed to be more glaring when the target population was adolescent girls coming from rural parts of West Bengal. So, one questionnaire was devised by the research team, with input from concerned persons, with the purpose of assessing adolescents’ knowledge and attitude pertaining to certain common mental health issues of their age. Before using this questionnaire in larger perspective, it was needed to validate this tool and to undertake one pilot study. This pilot study was conducted as a forerunner of another bigger study targeting around 1000-2000 adolescents meant to explore various mental health issues of that age group to formulate plans to help them cope up with those problems more effectively.


The aims of this current study were:

a. To prepare a Bengali questionnaire to assess knowledge and attitude of adolescents about the various mental health issues related to adolescence and validate it.

b. To conduct a pilot study with this questionnaire on adolescent school girls from rural background of West Bengal.


a. It was a cross sectional study conducted on 107 adolescent school girls from rural background of West Bengal.

b. Inclusion criteria

i. 12 to 18 years old school going girls

ii. Able to read, write and understand Bengali

iii. Informed consent from both the girl and accompanying teacher

c. Exclusion criteria

i. Poor comprehension of Bengali

ii. Disability hindering reading, writing or hearing

iii. Below average comprehension as assessed on clinical interview

iv. Unwilling to participate in study


A data sheet specially devised for this study containing certain personal data (like age, class etc) and Mental Health Awareness Questionnaire.

Mental Health Awareness Questionnaire: Psychiatrists, counsellors posted in Child and Adolescent guidance clinic, school teachers who watch child and adolescent population closely and a few persons who have adolescent children were approached by the research team, explained about the project and were requested to submit sample questions (in Bengali) pertaining to mental health issues of adolescents based on their day to day experience. Then, based on opinions of senior psychiatrists who attend to child and adolescent population in the OPD regularly, 20 questions from this list were selected and a questionnaire was constructed in Bengali. Then it was presented to the two counsellors posted in Child and Adolescent guidance clinic in the hospital, school teachers and parents of adolescents. Based on the consensus 13 questions were retained in the questionnaire. Out of these XIII questions, XII have multiple options to choose from and the last one is open ended. Some of the questions are case vignettes presented in simple words depicting certain situation, followed by choices. Out of these first XII questions, four (no. I,II,VIIandIX) are meant to assess knowledge about mental health issues in adolescence, while eight questions are kept observing their attitude towards some real-life examples related to mental health issues. The subjects are required to tick one choice for each of the first XII question that they think right. In the XIIIth question they are required to note down up to three ways they think that could help them feel happy.

For each of the knowledge questions one choice is correct and the frequency of correct answers is seen. For the attitude questions the frequency of the choices used by the subjects are observed. And for the open-ended question the ideas expressed by the subjects are noted.

Conduction of the Study

This pilot study was conducted after validating the questionnaire and after obtaining approval from the ethical committee (Ethical committee of Burdwan Medical College and Hospital). The purpose of the study was explained to the students and their accompanying teacher. Subjects who met the inclusion criteria were taken up in the study. The 13 item Bengali questionnaire were given to the girls, individually, after explaining the procedure to respond to it, before they attended the lecture. They were requested to complete the questionnaire on their own, unaided. Most of the girls returned the completed questionnaire within 30 minutes. After rejecting two data for multiple response in a few questions (from no. I to XII) and one data for not being returned, finally 107 samples were retained in this pilot study.

Validation of the Questionnaire: Face validity was determined through group discussion by experts

Test-retest reliability was seen by administering the questionnaire on a group of 20 girls of class VIII from a rural school nearby, twice, after a gap of 10 days.

Statistics: Statistical Package for Social Sciences (SPSS), 16th version, was used for the analysis of data.

Validation: Test-retest reliability was seen by kappa statistics.

Pilot Study: In the pilot study the distribution of age, class and religion in the sample was analyzed using descriptive statistics. For the knowledge-questions the frequency of correct response was seen by descriptive statistics. For the attitude-questions the frequency of using the choices in each question was noted through descriptive statistics. For the last question, i.e., the open ended one, subjects expressed several ideas about how to feel happy. So, the statements were grouped into eight broad categories, after reaching a consensus by experts. The frequency of responses in each category was seen by descriptive statistics.


Table 1: Test re-test reliability.


Test Re-Test Reliability: Eight out of twelve questions from I to XII had kappa value more than 0.7 indicating good test re-test reliability (Table 1). Questions II (0.875), 3 (0.861), 9 (0.886) and 11 (0.914) showed excellent test re-test reliability. Two questions had kappa value slightly less than 0.7 (questions 4 and 5), while the value was not satisfactory for questions VII (0.596) and 12 (0.490). In question no XIII most of the categories showed good to excellent test re-test reliability, though the category ‘no response’ had poor kappa value (0.200).

Table 2: Particulars of the sample.


Table 3: Response to knowledge questions.


Pilot Study: The mean age of our study population was found to be 13.11 (±0.91) years, majority of the girls were Hindu (69.2%) and were studying in class VIII (67.3%) (Table 2). While observing the response for the questions pertaining to knowledge (Table 3) it was found that there was more correct response in questions I (50.5%), II (52.3%) and VII (50.5%), though majority gave incorrect responses to question 9 (72%). Only 0.9% of the study population refrained from responding to each of the questions I and IX, while for each of the questions II and VII it was 4.7%. Regarding the questions pertaining to attitude (Table 4), question no X was attempted by all the subjects. 13.1% of the girls did not respond to question no 6, though this number was much less for rest of the questions (from 0.9% to 4.7%). For most of the questions the subjects preferred one response over others, more so in questions IV (86% choice 1) and XII (81.3%). In the open-ended question most of the girls came out with ideas about how to feel happy (question XIII) (Table 5). Most of their ideas were related to categories ‘sharing’ (52.3%), ‘sports’ (43%) and ‘food’ (39.3%). 9.3% of them refrained from responding to this particular question.

Table 4: Response to Attitude questions.


Table 5: Response to the Open-ended question (question no. 13).



Opinion from different quarter of persons and professionals who deal with adolescents regularly ensured that the questionnaire remains relevant to the target population, as well as, to the culture. Since knowledge and attitude about a problem influence a person’s behavior towards it, questions pertaining to both domains were kept in the questionnaire. Having knowledge about adolescents’ view of wellness and the ways to it might help us while dealing with their mental health issues [7]. So one open ended question was kept knowing about the ways, they think, could make them feel happy. A few questions were kept pertaining to stress, depression, anxiety and self-harm based on experience from previous studies [2,3,6,7]. Since majority of the school girls from this part of rural Bengal use Bengali as the primary language of communication the questionnaire was composed in Bengali. The fact that most of the questions had satisfactory to excellent kappa values demonstrates its reliability. Also, categorization of the responses to the openended question stands test of reliability. Poor kappa value of the ‘no response’ category (question XIII) might have resulted from the fact that some of the girls who refrained from responding to this question on first instance responded to it when presented the second time. Acceptability and feasibility were good as none of the subjects refused to respond to the questionnaire and most of them responded to almost all the questions unaided. From results it could be observed that though most of the girls answered the questions pertaining to what is meant by adolescence, mood swings during adolescence and cause of mental illness correctly, the difference between frequency of correct and incorrect responses was very less. Regarding question no IX where common depressive symptoms are elaborated, a little more than one fourth of the subjects could recognize those to be part of mental illness. Most of them thought these to be arising from reckless lifestyle or some physical illness. Coming to the questions related to attitude, in the question (no III) that raises the issue of mood swings, most of the girls think that the problem is serious, though next in number are those who think this could be overcome with help of close ones. While on direct question a good number of the subjects failed to recognize depressive symptoms as psychological in origin (question IX), on presenting case vignettes most of them readily identified the problem behaviors and came out with helping attitude (questions IV,V,VI and VIII). This seems to be in concert with previous observations where, despite a lack of knowledge about mental illness, most of participants expressed helping attitude. In the question pertaining to self-harm (question V) most of the girls thought this to be problem behavior, though quite a few of them think this to be a way of expressing grievances against parents [12]. It was also observed that most of them perceive their parents to be understanding and supportive and would seek help from them when faced with problems that they perceive as illness (questions X and XII). Though, while faced with academic setback most of the participants preferred to overcome the stressor themselves or to seek support from friends (question XI). This observation supports the findings of studies by where the subjects chose parents and friends as first point of contact to seek help [1,2,12].

Thus, it appears that adolescent girls, though have limited knowledge about adolescence, about mental health issues related to this period, they do identify when someone is in trouble and expressed readiness to help. They expressed reliance and grievances about their relationship with parents, and at the same time wanted to seek help from parents and friends when faced with some problem. This observation could be explained in the light of theories dealing with conflicts of adolescents [13]. This comfort zone with near and dear ones was further emphasized in their response about how to remain happy (question XIII). Most of them thought that sharing worries with friends, parents and others they feel close to; not worrying too much and resilience (‘remaining calm in face of problems’, ‘mental strength’ etc) could help them to be happy. Those observations highlight some of the aspects of positive mental health [2]. Though insignificant from statistical point of view, yet quite striking an observation was that two of the girls thought one can be happy if he/she can love others. A good number of opinions came in favour of games and sports as a way to remain happy. While a conspicuous number of the participants thought food can make them happy. This could be explained by the construct of ‘Societal indicators of positive mental health’ that mentions ‘adequate food’ along with some other factors [2].


This questionnaire appears to be a reliable tool to assess knowledge and attitude of our target population (adolescent school girls from rural background) regarding various common mental health issues related to adolescence. From the pilot study it could be concluded that acceptability and feasibility of this tool is satisfactory. Based on the responses it appears that there is a need to discuss various mental health issues related to adolescence to this population. This study exposed some the strengths of our adolescent population, like, an attitude to help and a sense of cohesion, as well as some of their concepts of remaining happy. The current study population did not include males, as well as, girls who were not going to school. Before generalizing the findings of this pilot study on adolescents larger and more inclusive studies need to be conducted.

Adolescents’ Mental Health Awareness Questionnaire


Age (years)


Please tick the correct answer of the following questions

I. Adolescence is the period between:

Youth and old age

Childhood and old age

Childhood and youth

II. Being listless, restless, losing temper and feeling guilt for no apparent reason during this period

Is usual

Should not happen

Happens with some but not with others

III. 3The problem mentioned in the previous question Is serious 1
Nothing could be done about this
Pass away uneventfully with hel of friends and near and dear ones 2
IV. If someone starts having adolescence retaed problems One should try to help him out 1
Avoid him
Avoid discussing the topic with him
V. Priyanka has consumed poison out of anger after being scoled at home. Her behaviour is
Abnormal 1
An ideal way to teach her parents a lesson 2
An attempt to commit suicide
VI. After failure in exam your classmate partha has been remaining absent in class for last 3 months. You have come to know that he is depressed. You think:
Since he has failed heshouldnt come to school 2
Depreesion is a mental illness that needs to be treted 1
His absence is ue to faile. He will become alright without any help.
VII. Wat, do you think causes mental illness
Influence of spirit and supernatural elements
Bad karma of past life
Poor hygiene
Both environmental and hereditary causes
VIII. Your best friend Uma has become withdrawn, remaing absent in class frequently and has become irritable for quite some time. You think
It’s wise to avoid her
You should complain to teacher against Uma. 2
She is doing this intentionally
She needs help. You should come forward 1
IX. Reduced sleep and appetite, lack of concentration and excessive worries for a long time is a sign of:
Physical illness
Mental illness
Is normal
Undisciplined life
X. In case you are observing those symptoms mentioned in the previous question in yourself for last one month, you will seek help from:
Parents 1
Doctor 4
XI. You are upset because of poor performance in exam, in spite of studying hard. You feel unable to speak out your distress. You think you should:
Hide your suffering from others
Seek help and support from friends 2
Should leave home
Should try to calm down through relaxation 4
XII. How, would you like to describe your relationship with parents:
They are understanding and supportive 1
They are supportive, though do not understand you
They do understand you, though do not cooperate
Always oppose you
XIII. What, you think, are the ways to remain happy?

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Lupine Publishers | Correlation between Ultrasound Findings of Ectopic Pregnancy And Operative Findings in Sudanese Women

 Lupine Publishers | Journal of Gynaecology and Women's Healthcare


Designs and objectives This study is a prospective, descriptive, hospital-based study, done in Omdurman Maternity Hospital and Omdurman New Hospital, to assess correlation between ultrasound findings of ectopic pregnancy and operative findings in Sudanese women.

Methods: The study was conducted on patients attended to gynecological Casualty with vaginal bleeding and abdominal pain in early pregnancy diagnosed as Ectopic pregnancy, ultrasound done to all the patients, with direct interview using predesigned questionnaire, and all of these is after consent obtained, ultrasonic findings was correlated to the intraoperative findings, sensitivity of the ultrasound was calculated using SPSS.

Results: out of 110 women, the most common age was 20-30 years (48.2%), multiparous were predominated by (53.6%). The commonest risk factors included; history of IUCD usage found in (52.7%), history of PID in (29.1%), history of abdomino-pelvic surgery in (24.5%), and history of ectopic pregnancy in (6.4%). On examination, there were (71.8%) women were stable, abdominal tenderness found in (54.5%), pallor in (57.3%), and (10%) of women were shocked. Sensitivity of U/S in diagnosis of ectopic pregnancy was 97.3%.

Conclusion: It was concluded that, U/S is reliable tool for diagnosing ectopic pregnancy

Keywords: Ectopic Pregnancy; Ultrasound Intraoperative; Sudan

Abbrevations: TVUS: Transvaginal Ultrasound; TAUS: Transabdominal Ultrasound; SPPS: Statistical Package for Social Science


Implantation of pregnancy outside the normal endometrial cavity called an ectopic pregnancy, which accounts for about 10% of all pregnancy-related deaths, despite improved diagnostic methods leading to earlier detection and effective treatment [1]. Also, it increases the chances of secondary infertility as well as incidence of the subsequent ectopic pregnancy [2]. The percentages of the women with ectopic pregnancy go to an emergency department with first trimester bleeding, pain, or both about (6 to 16%) [3]. Which is strongly associated with an increased incidence of pelvic inflammatory disease [4]. Symptoms may present in both ruptured and enraptured cases [5]. Ectopic pregnancy should be suspected in any woman of reproductive age with these symptoms, especially those who have risk factors for an extrauterine pregnancy [6]. The diagnosis is usually made clinically, based upon results of the imaging studies (ultrasound) and laboratory tests (hCG). The diagnosis can also be made by observation of the ectopic gestation at surgery or histopathological examination [7]. Transvaginal ultrasound (TVUS) is the principle approach used for sonographic evaluation of pregnancy of unknown location. TVUS allows for earlier and more reliable detection of an intrauterine or ectopic pregnancy (abdominal pregnancies are an exception) and for more reliable detection of a fetal heartbeat compared with transabdominal ultrasound (TAUS) [7]. A pseudosac can be seen in up to 20 percent of women with an ectopic pregnancy [8]. An extraovarian adnexal mass, seen in 89 to 100 percent of women with ectopic pregnancy, is the most finding in tubal pregnancy [8-10].


This is a descriptive, prospective hospital-based study .It was conducted in Omdurman Maternity Hospital and Omdurman New Hospital. Sudan, during the period from August 2014 to Jan 2015 all women diagnosed clinically and by ultrasound with ectopic pregnancy attended study area were included in the study. Time-frame sample size was be taken in a period of six months from August 2014 to Jan 2015. Variables assessed were: sociodemographic data, previous ectopic pregnancy, contraceptive usage, tubal pathology and surgery, PID, clinical presentation, ultrasound findings and intra-operative findings. Intra-operative findings were correlated with clinical presentation, examination, and ultrasound findings. Sensitivity and specificity was calculated. The data was collected by direct interview using predesigned questionnaire. Abdominal ultrasound, and/or vaginal ultrasound were done to all the patients according to clinical presentation. Ultrasound was done by senior obstetrician and radiologist. The data was analyzed by computer program; statistical package for social science (SPPS), results presented in tables and graphs. The test of significance was be calculated by P value (0.05: 95% confidence). Written consent was obtained from SMSB provided to the hospital administration. Women consent was obtained verbally. Privacy of data collected was considered.


The study included 110 women diagnosed as ectopic pregnancy by ultrasound and clinically and they attended to OMH and ONH during the period from Aug. 2014 to Jan. 2015. The study aimed to correlate clinical presentation, and ultrasound findings versus intraoperative findings. Age distribution showed that, 53(48.2%) of women in the age group of 20-30 years, 45(40.9%) in the age group of 31-40 years, 9(8.2%) in the age group of 19 years or less, other socio demographic distribution of women in the study shown in Table 1. Obstetric history showed that, 58(52.7%) of women found to have history of IUCD usage, 32(29.1%) had history of PID, 27(24.5%) had history of abdominal-pelvic surgery, 7(6.4%) had history of ectopic pregnancy and 40 one woman had subfertility. No woman found to have IVF as shown in Figure 1. In addition to clinical examination, transvaginal scan for confirmation of the diagnosis was used in 74(67.3%) of women, and transabdominal scan was used in (32.7%) of women. Operators were distributed to 51(46.4%) radiologists and 59(53.6%) consultants as shown in Figures 2 & 3.

Figure 1: Risk factors distribution among Sudanese women diagnosed with ectopic pregnancy at OMH and ONH, Aug. 2014 - Jan. 2015

Figure 2: Amenorrhea in Sudanese women diagnosed with ectopic pregnancy at OMH and ONH, Aug. 2014 - Jan. 2015.

Figure 3: Method of diagnosing Sudanese women with ectopic pregnancy at OMH and ONH, Aug. 2014 - Jan. 2015.

Figure 4: Intra-operative findings in Sudanese women diagnosed with ectopic pregnancy at OMH and ONH, Aug. 2014 - Jan. 2015

Table 1:

Intra-operative findings showed intact ectopic pregnancy in 28(25.5%), ruptured ectopic was found in 79(71.8%) and negative laparotomy found in 3(2.7%) as shown in Figures 4 & 5. Specific findings showed that gestational with positive fetal cardiac activity in 10(9.1%) of women, gestational sac containing fetal pole and/or yolk sac in 13(11.8%). Non-specific findings of ultrasound imaging showed complex mass in 3(2.7%), complex mass with free fluid in 54(49.1%), free abdominal fluid in 0(9.1%) and pre-pelvic + complex mass with free fluid in 20(18.2%). All participants found managed surgically by laparotomy. Correlation between intraoperative and ultrasound findings showed significant association (P value = 0.002), 19(67.9% out of 28 specific findings) of women with intact ectopic showed specific finings in U/S, among women with ruptured ectopic (79), 76(96.2%) had non-specific U/S findings, while all women with negative laparotomy had non-specific U/S findings. Correlating intra-operative findings with tender abdomen and shocked patients showed no significant association (P value > 0.05).

Figure 5: Ultrasound findings in Sudanese women diagnosed with ectopic pregnancy at OMH and ONH, Aug. 2014 - Jan. 2015


This study carried out in OMH and ONH to assess correlation between ultrasound findings of ectopic pregnancy and operative findings in Sudanese women total number of patients was 110, age distribution showed that, 53(48.2%) of women had age of 20- 30 years, 45(40.9%) had age of 31-40 years, 9(8.2%) had age 19 years and less. Multiparous women predominated among women in the current study, they represented by (53.6%), on the other hand findings indicated considerable percentage of women who were grand multiparous (32.7%) versus few primigravida (13.6%). Multi-parity revealed by Kopani F and colleagues as risk factors for ectopic pregnancy 56. The most frequent criteria considered as risk factors for ectopic pregnancy in the current study were history of using intra-uterine contraceptive device, history of pelvic inflammatory disease and history of abdomino-pelvic surgery (previous caesarean section) and they were represented by 52.7%, 29.1% and 24.5% respectively. Other risk factors with less incidence were history of ectopic pregnancy (6.4%) and infertility. Similar findings were reported by Parashi S and colleagues in 2014, who revealed significant association between prior ectopic pregnancy, prior tubal ligation, use of intrauterine device, and prior abdominal/pelvic surgery with ectopic pregnancy (p<0.05) 57 (12).

Of the risk factors reported by Kopani F and colleagues, there was previous surgery was registered in 26% of patients and history of previous ectopic pregnancy in 7-8% of patients reported high incidence of ectopic pregnancy [5-7]. While other studies vast majority of women in the current study presented with vaginal bleeding and abdominal pain (86.4%), while abdominal pain was found in 13.6% women. These findings are compatible with what reported by Parashi S and colleagues [8] in 2014 who found that, initial symptoms were nonspecific, consisting of a period of amenorrhea and abdominal pain or tenderness, with or without unexpected vaginal bleeding. New onset pain was reported, dull or sharp in nature, which was generalized or localized to one area. Furthermore, they complained about spotty or irregular vaginal bleeding. Other study reported that, abdominal pain was a presenting symptom in 99 percent, amenorrhea in 74 percent, and vaginal bleeding in 56 percent. On examination at presentation, most patients found stable, pale and have abdominal tenderness which represented by 71.8%, 57.3% and 54.5% respectively. Shock was found in 10%, and no soft abdomen noticed. In absence of ultrasound findings, HCG test, clinical manifestation of ectopic pregnancy complicates the diagnosis because of their broad spectrum that run from asymptomatic until acute abdomen and hemodynamic shock.

Most women diagnosed through transvaginal scan (67.3%), less women diagnosed by transabdominal scan. Kopani F and colleagues revealed that, trans-vaginal ultrasound has a sensitivity of 97% and specificity of 95%. The most frequent intra-operative findings of the current study was ruptured ectopic (71.8%), followed by intact ectopic which found in 25.55% and negative laparotomy in 2.7%. Intra-operative findings were reported by Tulandi T showed that, in one representative series of 147 patients with ectopic pregnancy (78 percent were ruptured) [8]. On the other hand, most findings by ultrasound diagnosis were non-specific (79.1%) (E.g. Complex mass with free fluid and free abdominal fluid), while specific findings reported in 19.1% (e.g. Gestational sac containing fetal pole and/or yolk sac). An extra ovarian adnexal mass was reported in previous study as most common non-specific sonographic findings; they reported that, it was seen in 89 to 100 percent of women with ectopic pregnancy, is the most finding in tubal pregnancy (20,26). The sensitivity of U/S method in the current study was found to be 97.3%. Findings of the study revealed good accuracy of ultrasound method to diagnose ectopic pregnancy, because imaging was mostly done by seniors and any suspicious result repeated several times in other place.

Out of 110 cases suspected with ectopic pregnancy with U/S method, only 3 cases found negative. Of the limitations faced by the current study, is scarcity of published literature comparing U/S imaging and intra- operative diagnosis in ectopic pregnancy patients, the only study available conducted in London by Condous G and colleagues in 2005, it assessed accuracy of TVS accuracy, and it was concluded that, the sensitivity of TVS found to be 90.9% . All women in the current study managed surgically by laparotomy, and they were all ectopic, salpingectomy was done. Intra-operative findings indicated that, intact ectopic were 28, out of which 67.9% showed specific findings in U/S. out of 79 ruptured ectopic, most cases showed non-specific findings in U/S. while all negative laparotomy showed non-Specific findings. Intra-operative findings indicated that, all shocked women in the study were found ruptured ectopic, except one case which was found intact. Correlating intra-operative findings with shocked cases and with abdomen tenderness found statistically not significant (P value was 0.326 and 0.230 respectively).


According to the current findings, we can conclude that, assessing the role of ultrasound in diagnosing ectopic pregnancy in comparison to intra-operative findings, high diagnostic accuracy was revealed with sensitivity of 97.3%. The study obtained good results because imaging was mostly done by seniors and any suspicious result repeated several times in other place. With regard to clinical presentation, most patients were stable, pale and have abdominal tenderness in addition to that some patients were in shocked. From the study the risk factors for ectopic pregnancy were history of using intra-uterine contraceptive device, history of pelvic inflammatory disease and history of abdominal-pelvic surgery. The most common intra-operative findings of the current study was ruptured ectopic, followed by intact ectopic and negative laparotomy.


a) Since ectopic pregnancy is a top emergency situation, and it is a leading cause of maternal death, clinical assessment should be done as accurate as possible, and cases should be scanned by highly skilled cadres.

b) Establishment of early pregnancy assessment unit in every maternity hospital.

c) Organization of IUCD workshops including proper treatment of associated infection.

d) All hospitals, especially obstetrical department should be supplied by well-established U/S unit and well trained operators.

e) Availability of laparoscopic unit with experienced personnel for managing intact ectopic and stable patients laparoscopically.

f) Ultrasound and intra-operative findings can help establishing a reported program to monitor, evaluate and manage ectopic pregnancy by routine studies.

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Wednesday, 28 July 2021

Lupine Publishers| The Relationship of the Trigemino-Cardiac Reflex to Sleep Bruxism

  Lupine Publishers| Online Journal of Neurology and Brain Disorders (OJNBD)


The trigemino-cardiac reflex is well researched in medicine. A newly proposed classification based upon the region stimulated elucidates sleep bruxism’s paradoxical effect on the trigemino-cardiac reflex, characterized by tachycardia, hyperpnea and hypertension. This article discusses this complex relationship and the resulting signs and symptoms reported in sleep bruxism, and how it meets these new criterion.

Keywords: Trigemino-cardiac reflex; Trigeminal cardiac reflex; Masseter inhibitory reflex; Sleep bruxism

Abbreviations: TCR: Trigemino Cardiac Reflex; MIR: Masseter Inhibitory Reflex; SB: Sleep Bruxism; GG: Gasserion (trigeminal) Ganglion; GERD: Gastro-Esophageal Reflux Disorder; OSA: Obstructive Sleep Apnea; RF: Reticular Formation; TMD: Temporomandibular Dysfunction; HR: Heart Rate (pulse); MABP Maximum Arterial Blood Pressure; 5-HT: Serotonin


The trigemino-cardiac reflex (TCR) is a unique and powerful brainstem reflex that has received a great deal of research interest. Sleep bruxism (SB) is sleep disorder that affects the TCR as well as other brainstem reflexes via stimulation of the brainstem; at the level of the gasserion ganglion (GG). This paper will discuss the unusual relationship of the TCR and SB in addition to how well sleep bruxism meets a new proposed classification system for TCR activation.

Literary Research

Relevant literature was identified through searching PubMed; Research Gate; Google scholar database and Mendeley web library using the search terms “trigemino-cardiac reflex”; “sleep bruxism”; “GERD”; and “masseter inhibitory reflex”.


Sleep disorders are an increasing health problem in all countries that have a direct effect on quality of life and productivity/safety of workers [1-3]. Sleep bruxism (SB) is a movement type sleep disorder characterized by transient tachycardia; tachypnea and hypertension occurring slightly before; during or slightly after the bruxism event; resolving immediately after the event ceases [4].

This increase in sympathetic activity has been shown to result from stimulation of the TCR at the level of the gasserion ganglion (GGa) [5-7]. SB is seen to occur concurrently with sleep apnea; occurring immediately before or after apnea events; but may also be seen on EMG studies independently of OSA [8]. SB also results in micro-arousals from sleep; classifying it as a true sleep disorder. Epworth Sleepiness Scale scores of 4 to 9 are characteristic of SB whereas scores of 10 and higher are suggestive of OSA4. The daytime sleepiness (males) and tiredness (females) seen in SB have similar deleterious effects on alertness; productivity and quality of life to OSA. In addition; SB is associated with the inception of chronic myofascial pain [9] affecting the orofacial region; tension and migraine type headaches; and temporomandibular dysfunction syndrome (TMD) affecting the temporomandibular joints [9-10].

The TCR is a powerful brainstem reflex that manifests as a sudden onset of hemodynamic influences on heart rate (HR); blood pressure (MABP) and has been associated with cardiac arrhythmias; asystole; apnea and gastric mobility [11]. It is an oxygen-conserving reflex that was first discovered in 1999 [12]; with considerable research ensuing [13-17]. The reflex may be activated by mechanical or chemical stimulation of the trigeminal nerve at any course along its distribution. Stimulation of the TCR results in neuronal signals being transmitted via the trigeminal nerve to the GGa; continuing to the sensory nucleus of the trigeminal nerve (V5) in the brain stem (mesencephalic nucleus). Signals are then transmitted polysynaptically through the reticular formation (RF); via short internucial fibers; to the dorsal motor nucleus of the vagus nerve (X). This pathway is considered as an afferent to the TCR (Figures 1-3). Parasympathetic neurons comprise much of reflex; arising in the motor nucleus of V5. Stimulation of V5 results in bradycardia; hypotension; as well as apnea and gastric hypermobility [17]. The reflex is of utmost importance during surgical procedures adjacent to the branches of the trigeminal nerve as the TCR can inadvertently be stimulated compromising the surgical procedure [18-21]. In procedures near or in the GGa; the opposite effect may be encountered: tachycardia; tachypnea and hypertension and gastric hypomobility [22,23].

Figure 1:

Figure 2:

Figure 3:

Meuwly; Galoanov et al have proposed a new classification for the TCR based upon where the trigeminal nerve is stimulated (Table 1) [17]. The classic “diver’s reflex” results from stimulation at the level of the first branch of the trigeminal nerve and the typical TCR response of bradycardia; hypotension and hypopnea. It is a cutaneous stimulation of the trigeminal nerve; on the ocular and nasal skin regions. The peripheral classification includes the actual orbit of the eye (by compressing the orbit); the maxillary branch (V2) and the mandibular branch (V3). Only the maxillary and mandibular stimulation regions result in the paradoxical sympathetic response of tachycardia; tachypnea and hypertension (Figures 1-3). In this classification; the only region of stimulation of the TCR occurs at the level of the GGa; all other regions react as expected. This stimulation at the level of the Gasserion ganglion can result in paroxysmal brady or tachycardia; hyper or hypotension and apnea or hyperpnea; depending upon the stimulus. A recent study described this phenomenon and concluded from the research that HR was the most significantly affected and the most useful measurement in the determination of TCR [24]. A diagnostic criterion that is generally accepted is of a heart rate decrease (or increase) of 20% or more from baseline; for a positive TCR diagnosis [17]. Table 2 lists other cause-effect relationship criterion proposed to diagnose and differentiate TCR proposed by Meuwly et al [17]. Sleep bruxism meets the first five of seven criteria of Table 2. GGa nerve blocks have an elevated risk profile and do not meet ethical standards for trials; and anticholinergic drugs fail to suppress the TGR effects of SB. By utilizing this additional criterion as well as the decrease (or increase in the case of SB) of HR by greater than 20% ; discrimination of other sympathetic and parasympathetic disorders that could affect HR and mimic the TCR is possible; rendering a more accurate diagnosis of this condition [24].

Table 1: The Extended Classification of the Trigeminocardiac Reflex.

Table 2: Additional Criterion based upon Cause and Effect (2 Positive Criterion = Positive TCR Stimulation).

Research has also found a genetic commonalty in SB sufferers: a polymorphism of the HTR2A gene on chromosome [13-25,26]. This gene codes for 5-HT (serotonin) receptors in the brain and gut. The masseter inhibitory reflex (MIR); another brainstem reflex; is located in the mesencephalic nucleus and has the MIR reflex nucleus affected by this polymorphism [27,28]. The resulting hypersensitivity to serotonin results in loss of inhibition by the MIR as well as activation of central pattern generators involved with chewing. In SB; extreme muscle contractions of the masseter; temporalis and suprahyoid muscle groups result [29]. The HTR2A polymorphism; seen in SB; was found to occur frequently in other conditions including obstructive sleep apnea; tension headaches; migraine headaches (without auras) and a myriad of psychiatric disorders [30-33]. Interestingly; OSA has headaches as a commonly reported symptom; often upon waking (assumed to be due to hypercapnia). In recent studies of the TCR it was demonstrated that; at the connection at the level of the reticular formation and nucleus ambiguus; there appeared to be an endogenous modulation. It was also found that the 5-HTR1A and 5-HTR2A serotonin reception genes were involved in mediation (stimulation; depression) of the connections (antagonists altered the TCR) [17]. With the 5-HTR2A polymorphism known to exist in SB; it is reasonable to assume that this and other regions could also be similarly affected (potentially resulting in hypersensitivity of the TCR).

During SB bursts; there is an ensuing cascade of events that occurs (Table 3). There are a number of cranial reflexes involved including the TCR; the MIR; the sucking reflex 4 (which can result in trauma of the buccal mucosa and development of linea alba lesions); and the swallowing reflex [34-36]. Scalloped borders of the tongue are also a common finding in SB; resulting from activation of the genioglossus muscle pressing the tongue forcibly against the teeth [37,38]. In other research it has been shown that there is a commonalty between SB and gastro-esophageal reflux disorder (GERD); supporting SB’s influence on the vagus nerve [39,40]. This can manifest as tooth erosions on the inner or lingual surfaces of the teeth and increase susceptibility to dental caries [41].

Table 3: The Sleep Bruxism Cascade.

With the suppression of the MIR; the forces generated during SB events far exceed those of normal chewing [29]. Damage to the teeth is common including:

a) Sensitive; loose or broken teeth [44]

b) Abfraction lesions [44]

c) Accelerated periodontal disease [45]

d) Formation of mandibular tori [46]

e) Eagle’s syndrome (calcification of the stylohyoid ligaments) [47]

f) Elongation of the coronoid processes [48]

g) Excessive compression of the TMJ; often initiating or accelerating degenerative changes [49-51].

As expected; these forces can be a contraindication to some dental procedures including porcelain crowns and inlays [52,53] and dental implants [54,55].


When considering how SB affects the TCR and MIR; the range of signs and symptoms observed during SB events can be readily interpreted. Restless leg syndrome (RLS) is another movement type sleep disorder that results in activation of the TCR similar to SB. Tachycardia; hyperpnea and hypertension (TCR stimulation); shown to also occur in RLS; has been shown to be a risk factor for heart disease in a number of studies [56,57]. To date SB has not been studied to the extent of RLS and it is not known at this time if SB is also a risk factor for heart disease. With the significant effect of SB on the TCR; Further research is certainly warranted.

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Monday, 26 July 2021

Lupine Publishers| Is Treatment Resistant Focal Epilepsy Less Frequent in Veterans?

 Lupine Publishers| Online Journal of Neurology and Brain Disorders (OJNBD)


Rationale and objective: Epileptic seizure disorders have become an increased source of concern in veterans given their relative high exposure to traumatic brain injury (TBI). 40% of adults with focal epilepsy are expected to develop treatment-resistant epilepsy (TRE), which can be amenable to treatment with epilepsy surgery. Yet, epilepsy surgery in veterans with epilepsy (VWE) is performed less frequently than in non-veterans with epilepsy. One possible explanation may be that when seizures begin after the age of 50, seizure freedom is likely to occur in 70% of patients. The purpose of this study was to examine whether the frequency of treatment-resistant epilepsy was different in veterans and to identify potential variables that may account for this difference.

Methods: In this retrospective study we included 157 veterans followed in the outpatient clinic of the Miami Epilepsy Center of Excellence Veterans Health Administration. Data collected from the medical records included age at onset of epilepsy, etiology, seizure type and epilepsy syndrome, response to pharmacotherapy, presence of psychiatric co morbidities (classified as mood disorders, generalized anxiety disorder, post-traumatic stress disorder, polysubstance abuse and other), antiepileptic regimen and adherence to medical treatment.

Results: Among the 157 patients, the mean age was 56.7 (±15.4) years and 140 (88.5%) were males; 119 patients (75.7%) had focal epilepsy presenting with complex partial with or without secondarily generalized tonic-clonic (GTC) seizures. TRE was identified in 25 patients (15.9%; 95% confidence interval: 11.0% to 22.5%); being a woman (p<0.01) and having focal epilepsy (p=0.04) were the only two significant variables associated with the development TRE.

Conclusion: In this study, the prevalence of TRE in this cohort of veterans was lower than that reported in the general epilepsy population. These findings need to be replicated in a larger study that includes the 16 VA Epilepsy Centers of Excellence.

Keywords: Focal epilepsy; Treatment resistant epilepsy; Major depressive episode; Traumatic brain injury; Posttraumatic stress disorder

Abbrevations: TBI: Traumatic Brain Injury; TRE: Treatment Resistant Epilepsy; VWE:Veterans with Epilepsy; GTC: Generalized Tonic Clonic; ILAE: International League against Epilepsy; PWE: Patients with Epilepsy; AEDs: Antiepileptic Drugs; PTSD: Post Traumatic Stress Disorder


Epileptic disorders have become an increased source of concern in veterans given the relative high exposure to traumatic brain injury (TBI), particularly because of the increased number of veterans who have suffered from serious TBI in the course of the wars in Aphganistan and Iraq. Furthermore, TBI can often result in the development of treatment-resistant focal epilepsy (TRE), for which epilepsy surgery can at times be one of the potential treatments. Yet, the use of epilepsy surgery among the 16 VA Epilepsy Centers of Excellence during fiscal year 2016 revealed only 8 surgical procedures in veterans with epilepsy (VWE) among a total of 5.980 unique patients seen during 2016 [1]. The purpose of this study was to investigate whether TRE in VWE followed at one Epilepsy Center of Excellence differed from that reported in the general population of patients with focal epilepsy and if so, to identify potential causes for such difference.

The international League against Epilepsy (ILAE) proposed the new definition of TRE as a failure to reach seizure remission after adequate trials of two tolerated, appropriately chosen and used antiepileptic drugs (AEDs) [2]. It is estimated that between 30% and 40% of patients with epilepsy (PWE) fail to reach seizure freedom, despite multiple trials with AEDs [3]. Several variables may contribute to the development of intractability, including lack of response to the first AED, specific syndromes, symptomatic etiology, family history of epilepsy, psychiatric co morbidity, high frequency of seizures, and early age at onset of epilepsy [4,5]. These observations suggest that prognosis of the seizure disorder can often be determined in the early stages of the disease. Recognition of these variables has a direct bearing on the management of these patients, as they may ensure an early referral for a pre-surgical evaluation and when possible, may shorten the medico-social and economic burden of intractable epilepsy [6]. On the other hand, some studies have suggested that late-onset epilepsy (beginning after the age of 60) may be more responsive to medical management than epilepsy diagnosed during adolescence and early adulthood [7-9].


This was a retrospective cross-sectional study. We reviewed the medical records of every consecutive patient with epilepsy followed at the outpatient epilepsy clinic of the Miami VA Healthcare System (VAHS) Epilepsy Clinic over a period of 24 months (January 2010 to January 2012). The study was approved by the Miami VAHS institutional review board. Two Board-certified epileptologists confirmed the diagnosis of epilepsy using the ILAE criteria (2010), based on the clinical history, neuro imaging data and electrographic recordings. The data extracted from the medical records included demographic variables (age, gender, ethnicity) epilepsy-related variables (seizure type(s) and epilepsy syndrome (focal or generalized), etiology of the seizure disorder (unknown, remote symptomatic [tumor, stroke, TBI, or “other”] or idiopathic), age of onset of epilepsy, co morbidities (medical, cognitive and psychiatric co-morbidities [classified as mood and /or anxiety disorder, post-traumatic stress disorder (PTSD), and polysubstance abuse/dependence, other] AED treatment, were recorded. We used the ILAE definition of TRE cited in our Introduction [2]. We defined epilepsy “in remission” as the absence of seizures in the last two years from the time the patient was evaluated at the epilepsy clinic. Of note, any patient with persistent seizures and/or in whom the clinical semiology of their seizures was not typical of an epileptic seizure underwent a diagnostic video-EEG monitoring study to establish if these events were epileptic or non-epileptic events and if epileptic seizures, to identify the type of seizures.

In this study 15 of the 25 patient with TRE underwent video- EEG monitoring, including all the women in this case series. The clinical characteristics of seizures of the 10 patients with TRE who did not have a video-EEG monitoring study were typical of epileptic seizures. Patients who were thought to have psychogenic non-epileptic seizures were excluded from the study. In addition, 9 patients who had persistent seizures but had only undergone one optimal trial with an AED were excluded. A total of 157 patients with epilepsy were included in this study. Demographic, epilepsy-related variables and data of psychiatric co morbidities were extracted from the medical record and included: age, gender, ethnicity, race, seizure type (focal, generalized), whether the seizure disorder was idiopathic, Symptomatic or unknown), age at onset of epilepsy, list of current AEDs and dosage (s), and response to each AED trial. Data of psychiatric co morbidities included mood disorder, anxiety disorder, post-traumatic stress disorder (PTSD), polysubstance abuse and other psychotic spectrum disorders such as schizophrenia, schizoaffective disorder and psychosis NOS.

Data Management and Analysis

Age was treated as a continuous variable and t tests for independent groups were used to compare data between TRE and seizure free group. Categorical variables were summarized by accumulated percentages. Chi-square or Fisher exact tests were used to compare the categorical variables for association between seizure freedom and clinical factors. Where the expected counts were less than 5, Fisher exact tests were performed. For all analyses, p≤ 0.05 was defined as statistically significant. Statistical analyses were performed using SAS software version 9.2 (SAS Institute, Cary, NC, USA).


Among the 157 patients included in our analysis 25 patients met criteria of TRE and 132 were seizure-free. (Table 1) summarizes the data pertaining to the demographic, epilepsy-related and co morbidities variables among the seizure-free and TRE patients. The prevalence of patients with TRE was lower (15.9%, 95% confidence interval (CI): 11.0% to 22.5%) than that reported in multiple studies, which ranges between 30% and 50%) (2, 10-12). Had we included in our analysis the 9 patients with persistent seizures that had been tried on one AED, the percentage of patients with TRE would have only increased to 21.6% (95% CI: 15.9% to 28.7%), still below the expected rate. Two variables were significantly associated with the development of TRE in our patients: female gender (47%) and focal epilepsy (92.0 %). See (Table 1) for statistical analysis.

Table 1: Characteristics of Study Cohort (N=157).

TRE: Treatment resistant epilepsy; SD: Standard deviation, GTC: Generalized tonic clonic

TBI: Traumatic brain injury, PTSD: Post traumatic stress disorder

Percentages are reported to one decimal digit


Epilepsy has become a neurologic disorder of great concern among veterans, given the large number of soldiers returning from the wars in the Middle-East where they suffered TBI [10]. This study was developed to assess the cross prevalence of TRE among outpatient veterans followed in a VA Epilepsy Center of Excellence and compare it to that published in populations of non-veteran PWE. To our surprise, the prevalence of TRE was lower in our patients than that published in studies conducted in non-veterans [11,12]. The second surprising finding was the observation that TRE was significantly more prevalent among women veterans. At this point, we cannot explain the reasons for a lower prevalence of TRE in our patients and our findings will need to be replicated in larger prospective studies. Of note, neither the cause of epilepsy, nor the age of onset of the seizure disorder failed to account for a better seizure control in our cohort, as suggested by several published studies that had suggested that elderly patients are more likely to have good outcomes [13,14].

In PWE, those with symptomatic or cryptogenic epilepsy are more likely to be medication resistant than those with idiopathic epilepsy [8,15]. In our study, the etiology of seizures did not play a factor in developing intractability. Traumatic brain injury was the most common cause of symptomatic epilepsy but failed to represent a predictive factor of intractability-perhaps because there are few cases of penetrating head injury, which is most commonly associated with medial intractability. Initial seizure frequency has been identified as a significant prognostic factor for TRE in the literature [16]. Unfortunately, we were not able to investigate this issue in a reliable manner because most of the patients were diagnosed somewhere else several years prior to our evaluation and the information was not found in the retrospective review of the medical records. This study has several limitations. First, this was a retrospective study. Second, we relied on the patients’ self (and or family members’) reports of their epileptic seizures. Yet, patients may have seizures and not be aware of their occurrence, which could result in an under-estimation of seizure frequency. Third, our study was based on data from a single Epilepsy Center of Excellence. While these are the centers where veterans with TRE are referred for treatment in the VA system, our data will need to be replicated in a larger study that includes all 16 Epilepsy Centers of Excellence in the USA.


The findings of this study may suggest that TRE may be less frequent in VWE than in other published cohorts and that women veterans may be at greater risk. These findings have to be considered as preliminary but deserve a careful investigation in a larger study that may include the other 15 Epilepsy Centers of Excellence of the VA and which can be compared with non-veteran populations.

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Lupine Publishers| Mini View on Current Trends in Computer Sciences & Applications

 Lupine Publishers| Current Trends in Computer Sciences & Applications (CTCSA)



Computer science has contributed a lot for making the life of human being smooth. The recent developments in the field of computer science are proven to be more smarter and more applicable structures result from marrying the learning capability of the applications with the transparency and accuracy. Foundations of computer science applications highlights the advantages of integration making it a valuable resource for the students and researchers in engineering, computer science and applied mathematics. The authors’ tried to lime light various applications that are an asset to industrial practitioners, corporates, academicians and professionals for control systems, data analysis and optimization tasks. With the continuous improvisation in the computer science applications the need of the young generation is fulfilled, and they can achieve their targets with the help of updated and enhanced support system. Authors are highlighting on current trends in computer sciences & applications and further illustrate how these various technologies integrate with social and economic factors to provide a thorough solution to the real-world problems of the human being in every domain of life.

The authors demonstrated how a combination of both techniques and human interventions enhances control, decision-making and data analysis systems.

Keywords: Computer; Trends; VLSI Technology; Multiprocessor; Parallelism; Configurable Computing; DSP; Internet


Although the very state forward answer for the latest trends in Computer science could be Machine learning, cloud computing and Artificial Intelligence. But basically, Industry build the software not only with what is new but by what customer problem can be solve easily and with good future scope and current market trends which covers customer requirements, this force towards innovation and create next generation products that can be quickly adopted for solving new use cases by Connecting to new data sources easily.

Top technologies which are in current trends in computer science & Application are as below:

A. Deep learning or Machine learning (ML).

B. Digital currencies: Example Bitcoin

C. Blockchain.

D. IoT

E. Robotics

F. Big Data Analytics.

G. Cloud Computing

H. Cyber Security

I. Virtual Reality

J. Predictive Analytics

The emerging areas that are seeking attention of many researchers in the field of computer science are designed and developed according to the latest market trends. Now a day trends in information technologies are directly or indirectly associated with the customer centric approach. One of the latest technology like computational biology where in the gathering and processing of biological data with the use of computer programs. This technology covers under Bio Informatics, which works with the combination of computers and living beings. It converts the biological data into readable format. This is helping the medical science a lot. Another most promising technology of today is Data Science or Big Data. This field has a very large and promising scope of research and development considering the huge volume of data being produced by organizations and individually in different sectors worldwide. It deals with the storage processing and analysing the massive data stored across the world in various organization and data centres.

The existence of newest trend of Virtual Reality cannot be ignored. The biggest stakeholders of VR applications are medical science, physical sciences, environment, businesses, space industry and entertainment industry. VR produces the set of the data which is used to develop new models training methods, communications and interaction. The major disadvantages in the use of VR application are time, cost and technological limitations. But because of its support system it is expected to become more affordable in future, today’s generation is grown up having technology at their disposal. They are familiar with smart phones, tablets therefore VR Developments will also increase in number of professionals more acquainted with the technology. Cloud Computing has already become the area of attention by most of the researcher and scientists. Cloud provider is basically data or internet provider. This plays an important role in various fields of business, computing security etc. This application works on the shared pools of configurable computer system recourses and higher-level services that ease the managerial effort with leads to economics of scale and development. It helps in running business more efficiently.

Cloud computing eliminates the capital expenses of buying hardware and software along with other related expenses. Business has become very flexible as cloud computing services are available on demand that leads to delivering right amount of IT resources resulting in scale elasticity. Lots of ‘Racking and Stacking’ task is being eliminated as cloud computing removes the needs for many of these tasks resulting in more time devotion towards more important business goals. Cloud computing services run on a worldwide network regularly upgraded data centres. This reduces network latency for application and improvises efficient computing hardware. It also helps in providing security to the data, apps from potential threats. However, several types of cloud computing is operational to help offer right solution for your needs like public, private and hybrid.

Deep Learning or Machine Learning is sub set of artificial intelligence and in today’s trends it’s one of most widely used computer science application, the ability of ML is to self-trend from data or able to learn from its own experiences, which can improve from application behaviour or experience without being explicitly programmed. Machine learning focuses on the development of computer programs that can access data and use it learn for themselves.

I. Example:

A good example can be a Navigation system or MAP application which initially developed with limited data but later on when this application gets used its design in a way so it trained itself to predict the best possible path.

Google search, uber, Pay Pal, Facebook are the good example of ML and these actually improving the usability of their services by applying deep learning algorithms. Below is the comparative example where one sector is using Machin learning and takin its benefits and other one is moving very slowly in digital and it’s far behind (Figure 1). Machine learning can help banks, insurers, and investors make smarter decisions in a number of different areas:

Figure 1:


a) Customer and Client Satisfaction: Machine learning helps financial services on below key points.

i. By analysing user activity.

ii. Smart machines can spot a potential account closure before it occurs.

b) Reacting to Market Trends: Another aspect can be cover by using a good ML algorithm which is generating the alerts or by preserving the trained to track trading volatility or manage wealth and assets on behalf of an investor.

c) Calculating Risk: Good ML algorithms can analyse datasets and based on the dataset (credit scores, spending patterns, financial data etc.) to accurately assess risk in both insurance underwriting and loan assessments, tailoring them to a specific customer profile.


Trends in Computer Sciences & Applications changed drastically the life of one and all. Be it a student learning or business corporate or any other professional, computer science and its applications are extending their updated support system to give more effective performance infrastructure in every sphere. Recent developments gives acceleration to the development of a Digital currency or digital money introduction in the form of digital, Blockchain a digital ledger in which transactions made in cryptocurrency. The contribution is endless and so the developments in this field are boundless.

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Saturday, 24 July 2021

Lupine Publishers | Liquid Chromatography-Mass Spectrometry and Its Applications: A Brief Review

 Lupine Publishers | Journal of Organic and Inorganic Sciences


The Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical technique with very high sensitivity and specificity. LC-MS is combination of Liquid Chromatography (LC) and Mass Spectrometry (MS). With the Liquid Chromatography (LC) the separation of components can be done and then the sample eluents from LC are transferred into Mass Spectrometry (MS) where the detection, identification and determination of masses of components can be done in presence of other components. LC-MS is used in determination, of pharmaceutical drug substances, intermediates and its related compounds for quantitative and qualitative purpose. LC-MS is used most significantly in in-vitro dissolution, bio-equivalence, bioavailability and metabolite studies. Also LC-MS is used in basic research, agrochemical, forensic laboratories and food industries .In this article principle of LC-MS, instrumentation and its applications are briefly discussed.

Keywords: Liquid chromatography (LC); Mass spectrometry (MS); High Performance Liquid Chromatography (HPLC); Liquid Chromatography-Mass Spectrometry (LC-MS)


Liquid Chromatography-Mass Spectrometry (LC-MS)

The High Performance Liquid chromatography (HPLC) is one of most common analytical technique used in pharmaceutical industry for determination and quantification of drug substances and its related substances. Due to high reproducibility and accuracy, HPLC is routinely used in pharmaceutical, chemical and pesticide industries.

The Liquid Chromatography-Mass Spectrometry (LC-MS) is hyphenated analytical technique which is combination of Liquid Chromatography (LC) and Mass Spectrometry (MS). HPLC (LC) separates the components of mixtures by passing through chromatographic column. Generally, the separated components cannot be positively identified LC alone. Mass Spectrometry is also used for identification of unknown compounds, known compounds and to elucidate the structure. Mass spectrometry is alone not good for identifying mixtures because mass spectrum mixture is actually complex of overlapping spectra from separated individual components. It is difficult to connect Liquid chromatography (LC) with Mass spectrometry (MS). An interface is used to transfer the liquid eluents from LC to MS.LC-MS is more significantly used in invite dissolution, bioavailability, bioequivalence and pharmacodynamics studies [1]. Preparative LC-MS systems can be used for rapid mass-directed purification of specific substances from such mixtures that are important in basic research, pharmaceutical, agrochemical, food and other industries [2,3].


Liquid chromatography-mass spectrometry (LC-MS)

The Liquid Chromatography-Mass Spectrometry (LC-MS) is combination of Liquid Chromatography and Mass Spectrometry which is used with separation power of HPLC with detection power of Mass Spectrometry (MS). The schematic block diagram of LC-MS is shown in below figure 1. The different parts of LC-MS instrument are listed as below.

    a. Liquid Chromatography (LC)

    b. Mass Spectrometry (MS)

Liquid Chromatography (HPLC): The Liquid Chromatography (LC) is a high performance liquid chromatography in which separation of components of mixture can be carried out by using liquid mobile and solid stationary phase. There are different types of chromatography like normal phase liquid chromatography, Reversed phase chromatography, Ion-exchange liquid chromatography, Chiral separation and affinity liquid chromatography [3]. By using different packing of columns with high efficiency small amount of complex mixture can be separated. The components of HPLC are listed below:

Figure 1: Schematic Block Diagram of LC-MS System.


    a. Pump: It consists of material which is inert towards solvents or any mixed composition of aqueous buffer and organic solvents. It delivers high volume of mobile phase up to 10mL/min. There are three major types of pumps are used i.e. reciprocating pump, Syringe pumps and constant pressure pumps.

    b. Sample injector: It is used to introduce sample volume into the chromatographic system. Generally sample volume from 1|iL to 100|iL can be injected. The injection volume can be increase by injector loop up to 2mL volume. There are two major types of injectors used i.e. Automatic injectors and Manual injectors. Automatic injectors are more comfortable and user friendly and are more accurate and precise as compare to manual injectors [3].

    c. Columns: It is stationary phase which consists of silica material in combination with carbon chain. Generally the column length used is about 50mm to 300mm. The columns used in HPLC are consists of Octadecyl (C18), Octyl (C8), Cyano, Amino, Phenyl packing's. The columns are used on the basis of nature of compounds to be separated [4].

    d. Detectors and recorder: The detectors is most important part of HPLC .There are different types of detectors used are UV-Visible detectors, PDA detectors, Refractive index (RI) detectors, Electrochemical detector, Fluorescence detectors and conductivity detectors. The signal received from detector can be recorded as peak and respective data can be stored in a software.

Mass spectrometry: Mass Spectrometry is analytical technique based on the measurement of the mass to charge ratio of ionic species related to the analyte under the investigation.MS can be used to determine the molecular mass and elemental composition of an analyte as well as in depth structural elucidation of the analyte [5]. In LC-MS there are two key components, ionization source and Interfaces. Below listed are the different components of Mass spectrometers as below.

    a. Ionization Sources and Interfaces

    b. Mass Analysers

Ionization/Ion Source and Interfaces: The Liquid chromatography separates mixture of components which are in liquid form, usually contains methanol, acetonitrile and water. This liquid containing mixture of components is transferred into the ion source of mass spectrometer. As ion source is under high vacuum. Due to the difference in the pressure it is difficult to mass to vaporize the liquid drops without losing mixture of components. Hence interfaces are used to resolve this problem. The different types of interfaces commonly used in mass spectrometer are described as below.

    a. Direct liquid Introduction (DLI): The ionization in Direct Liquid Introduction (DLI) is generally accomplished by vaporizing solvent as a chemical ionization and reagent gas. Both the normal and reverse phase solvent system have been used. Reverse phase solvents used are methanol/water, acetonitrile/ water mixture up to 60% water. In general buffer with salts are not allowed as there is chance of capillaries to plug when heated.

The operation of Direct Liquid Introduction (DLI) is combination of thermal energy and liquid flow rate. The liquid enters the interface at limited flow rate only. The analyte ions produced with the help of thermal energy then transferred into ion source through capillary inlet or pinhole diaphragm [6,7].

    b. Atmospheric-Pressure Ionization (API): In Atmospheric-pressure ionization (API) contains three major steps i.e. Nebulisation, Evaporation and Ionization. There are two main modes of API are Electrospray Ionization (ESI) and Atmospheric-pressure ionization (APCI). In Atmospheric- pressure ionization (API), when stream of liquid (solvent) containing a sample is passed through narrow capillary tube and nebulized at large chamber, mist of small droplets is produced .The ionization process takes place and the proportion of droplets carry an excess of positive or negative electric charge .In large heating chamber the evaporation of solvent takes place. The solvent evaporates from the droplets to form smaller and smaller. The collision takes place between the molecules and ions. The resulting ions then passed through capillary into mass analyser [2,8].

The Atmospheric-pressure ionization (API) is technique used for wide range of polar and non-polar analytes of moderate molecular weights.

    c. Electrospray Ionization (ESI): The Electrospray Ionization (ESI) is most useful ion source developed by Fenn and his colleague's. In Electrospray Ionization (ESI) the liquid sample passed through a stain steel capillary tube which is maintained at high positive or negative electric potential about 3- 5kV [1]. Due to this the charged droplets are formed at the capillary tip which are then undergoes vaporization process. The solvent gets evaporated from droplets, and undergoes reduction in size and surface charge increases. The collision takes place until the highly charged droplets are converted into gas phase ions. These gas-phase ions pass through the capillary sampling orifice into the low pressure region of the ion source [9].

The major advantage of ESI is that the ions are multiply charged, the number of charges increased by 1 to 3 for a molecule 1000Da or above 50000Da. This yields an m/z ratio that is always below 2000. LC-MS with an Electro spray ionization (ESI) is used to measure the molecular weight of peptides, Proteins, Biological samples, Polymers, nucleotides, sugars and organometallics. It is also used frequently in Biological research and medical analysis [10]. The Schematic diagram of ESI is shown figure 2.

Figure 2: Electrospray ionization source.


    d. Atmospheric Pressure Chemical Ionization (APCI): The Atmospheric Pressure Chemical Ionization (APCI) include two major steps, evaporation /desolvations of analytes and charged transfer reaction in vapour phase to generate the vapour phase ions.

In Atmospheric Pressure Chemical Ionization (APCI) liquid (solvent) containing sample is nebulised through narrow capillary tube and nebulized into large chamber. In large heating chamber the evaporation of solvent takes place at atmospheric pressure and small droplets are produced. The ionization takes place. Generally ionization takes place at 250 to 400 °C. The ions are then transfer the charges to molecules through chemical reactions. The resulting ions are pass through capillary orifice of mass analyser. It is widely used for less polar and non-polar analytes having moderate molecular weights [11].

    e. Thermo spray and Plasma spray Ionization (TSPI): The Thermo spray is used as both liquid inlet system as well as ionization source. Plasma spray is modification of thermo spray.

In Thermo spray the liquid sample solution is passed through capillary tube which is heated and which causes the evaporation of solvent. The charged droplets are formed. Due to evaporation of solvent the droplets becomes smaller and smaller. The density of electric charge on the surface of droplets increases. The resulting ions are then passed into mass analyser with electrostatic voltage system [8].

The Plasma spray itself does not produce ions but the ions produce in thermo spray, with the help of corona discharge or plasma the number of ions can be increased. The electric discharge induces the more ionization in the neutral molecules. This enhancement increases the ionization of molecule. The plasma spray technique is more sensitive and it is widely used for analysis in clinical and medicine [12].

    f. Atmospheric pressure photo Ionization (APPI): In Atmospheric pressure photo Ionization (APPI), photons are used to excite and ionise the molecules. Atmospheric pressure photo Ionization (APPI) include mainly two steps i.e. excitation and ionisation of analyte from eluent.

Like atmospheric pressure chemical ionization (APCI) in Atmospheric pressure photo Ionization (APPI) the eluent from LC vaporize into gaseous phase. The APPI uses Kr lamp to produce photons. Kr lamp generates high energy photons which are used for excitation and ionization of molecules. The range of energy is selected to minimize the ionization of analytes. The ionized analytes are then transferred into capillary orifice into mass analyser (m/z).

This technique is useful for non-polar analytes which very much difficult to ionize with Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) [13,14].

g. Particle Beam Ionization: The Browner and his colleagues has developed particle beam interface to separate the solvent from solute with minimum loss of solutes. The nebulization and evaporation process are like Thermo spray (TSP), Atmospheric pressure chemical ionization (APCI), Electrospray ionization (ESI) [15].

In this liquid separated from HPLC or LC, eluent is passed through narrow tube. The liquid is injected with helium gas, due to this the spray of liquid droplets are formed with high velocity. The liquid drops from nebulizer passes through heating chamber, where the solvent begins to evaporate and liquid droplets becomes smaller and smaller. The spray of liquid droplets exits through heating chamber as a particle beam. Then this beam passes through ionization chamber similarly like Electro spray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) [2].

h. Continuous Flow Fast Atom Bombardment (FAB): The FAB is simple, high sensitivity technique of interface. In FAB liquid target is bombarded by fast atoms such as Argon(Ar) or xenon. The sample is dissolved in glycerol and spread on thin layer metal plate /probe. Then this probe is inserted into mass spectrometer and a beam of fast moving atoms bombards on probe and ionise the samples which then pass into mass analyser (m/z). FAB is used for large and thermally unstable molecules. It used for surfactants and proteins [16,17].

Mass Analyser: After ionization the ions are transferred into mass Analyser where the separation of ions are done according to their mass to charge (m/z) ratio. Generally mass Analyser used is on its speed, time, rate and its reaction.

Below are the mass Analyser:

    a. Quadrupole

    b. Time of flight

    c. Ion trap

    d. FTICR (Fourier transfer ion cyclotron resonance)

a. Quadrupole Mass Analyser: It is the most useful and commonly used mass Analyser. It consists of two plain of parallel rods which are located between an ion sources and a detector. The mass Analyser i.e. separation of ion according to their m/z in either time or space [6].

The linear Quadrupole mass Analyser consists of four hyperbolic or cylindrical rods that are placed parallel in a radial array. Opposite rods are charged is a +ve or -ve direct current(DC) potential at which an oscillating radio frequency alternating current (RF) voltage is superimposed [18].

The combination of DC and RF applied to the rods, trajectories of the ions of one particular m/z are stable these ions are transmitted towards detector. On the other hand ions of unstable m/z are discharged on the rods.

The ions introduced into Quadrupole by mean of low accelerating potential. The ions are oscillating in plane perpendicular to the rod length as they trends through Quadruple filter.

Ions of carrying m/z consequently be travelled towards detector by applying DC and RF voltage at constant ratio. The resolution depends on ratio of DC and RF potentials. Generally the Quadrupole is operated at <4000 m/z and scan speed up to 1000m/z passes. The unit mass resolution means that mass accurately is seldom better than 0.1 m/z [19].

The RF values are generally in the range 1-2MHZ. The DC Voltage may be 1000V and Maximum RF voltage is 6000v. The schematic diagram of quadruple mass analyser is shown below figure 3.

Figure 3: Schematic Diagram of Quadrupole Mass analyser. J


b. Time of Flight Analyser (TOF):

The time Flight is most robust used for wide variety of ions sources and inlet systems. In this is no any magnetic field, maintenance and calibration it is just simple electrostatic and straight forward. The ions are extracted from source and subjected to an accelerating voltage. The time taken to travel the length of the drift or flight to be depends upon the mass of ion and its charge [20]. For single charged ions (z=1, m/z =w) the time taken to reach the detector is proportional to mass of the ions. When the ions trends towards the detector the lighter ions will strikes the detector first [18].

Scanning of all the ions are detected simultaneously. The scanning the mass range is very rapid and can be used for very large m/z values. The schematic diagram of Time Flight mass analyser is shown below figure 4.

Figure 4: Time of flight mass Analyser.


c. Ions trap mass Analyser:

Ion trap mass Analyser is high resolution, high sensitivity and multiple product ion scan capability.

A Quadrupole ion trap is a three dimensional ion trap. It consists of cylindrical ring electrode to which Quadrupole field is applied. Another two are end capped electrodes [21].

One end cap electrode has single small central aperture through which electrons or ions are introduced into the trap while other one has several apertures or holes through which ions are passed to a detector. A Helium bath gas is present in the trap to stabilize the ion trajectories. The collision takes place between helium bath gas and ions. Due to this the motion of ions increases the trapping efficiency of analyser. The ions are ejected from the trap on the basis of mass to charge (m/z) values to create the mass spectrum [22]. The schematic diagram shown below figure 5.

Figure 5: Ion Trap mass Analyser.


d. Fourier Transfer Ion Cyclotron Resonance (FT-ICR):

The Fourier transfer ion cyclotron resonance (FT-ICR) is most important mass analyser. The ions arrived from ionization source are passed into mass analyser where they are separated according to their m/z ratio. The ions entered in chamber are trapped in circular orbits. The ions are accelerated by both electric field and magnetic field.

Due to this the ions get excited and generate time dependent current. The ions trapped separated according to mass to charge (m/z) ratios.


The detector is an important tool of mass spectrometer that produces the current that is proportional to the number of ions strike it. Once the ions are formed passed from analyser they have to be detected and transformed into signal. Below listed are the type of detectors commonly used.

Point Ion Collectors Detector:

In this the ions collectors are placed at fixed point in mass spectrometer. All the ions are focused upon the detector situated at single point. The arrivals of ions can be recorded by the flow of electric current and the data can be recorded. The electric current flow is proportional to the ions arriving at point ion detector.

Array Detector:

An Array detector is collection of point collectors placed in plane. The ions are arrived at a point or across the plane in array detector. The ions with mass to charge (m/z) values are separated and are recorded along plane using point ion collector. Spatially differentiated ions with the mass range are detected simultaneously at the same time in array detector [23,24] (Table 1).

Table 1: Comparison between LC, MS and LC-MS.



Application of LC/ESI-MS in In forensic sciences

LC-MS is used for determination of toxicity, in drug analysis and also in trace analysis. By using small amount of sample the toxins in different material can be determined with LC-MS. Any toxic metabolites in food or beverages can be determined by using LC-MS. E.g. Identification of detergent added into orange juice can be determined by analysing by the juice and detergent sample. The standard surfactant alkyl diphenylether sulphonic acid is used. Both juice and detergent samples are analysed in same chromatographic conditions. The mass chromatograms and mass spectra obtained from the juice and detergent samples are identical with the reference spectra of standard surfactant (alkyl diphenyl ethersulphonic acid) [25,26] (Figure 6).

Figure 6: Extracted Ion Chromatogram of Orange Juice using LC-MS using ESI in negative mode.


Figure 7: LC-MS Chromatogram in ESI (+) of Tuaminoheptane and 4-methyl-2-hexaneamine and mass spectra of 4-methyl-2- hexaneamine which shows pseudo molecular ions [M+H]+ at m/z 116 yield the prominent ion at m/z 57.


Application of LC-MS in Doping Test:

The LC/ESI-MS with positive mode can be used for detection in urine of 4-Methyl-2-hexaneamine doping agent. The urine samples are analysed with addition of internal standard of Tuaminoheptane. The suspected primary amine 4-methyl-2-hexaneamine, an analog contained in dietary supplement, to be the unknown compound. The standard used is 4-methyl-2-hexaneamine which exhibits two unresolved peaks at RT 3.43min and 3.78min. which are identical with those of unknown compound [26]. The single reaction monitoring (m/z 116-57) was specific for detection of 4-methyl-2- hexaneamine as shown below figure 7.

Other Applications:

In Pharmacokinetics:

LC-MS is used in the study of absorption, metabolism, and excretion of drugs. Bio analytical methods are used for quantitative and structural elucidation of drugs and its metabolites in the biological samples (plasma, urine, saliva, serum etc.) [25].

In Bioavailability and Bioequivalence study:

Comparative bioequivalence studies in which quantitative determination of drugs or metabolites is measured in biological matrix, pharmacodynamics, clinical trials and In-vitro dissolution tests [27,28].

In determination of molecular weights:

LC-MS is used for determination of molecular weights of known and unknown compounds. It provides the information about molecular weight, structure, identification, quantity of sample components. LC-MS is used for determination of molecular masses of proteins, nucleic acids, polymers and peptides.

In determination of Assay of drug and intermediates:

LC-MS is used in pharmaceutical industry for determination of assay of drug substances, drug products, intermediates and their related compounds [3].

In Agrochemical and pesticides industry:

It is used in determination of different components present in the fertilizers and pesticides [25].

Environmental Applications:

LC-MS is used for detection of phenyl urea herbicides, detection of low level of carbaryl in food [29].

Literature Survey:

Perrenoud L. developed LC-MS method for detection of 4- methyl-2-hexaneamine a doping agent from urine.LC-MS with ESI in positive mode is used. The analyte separated by gradient mobile phase on reverse phase C8 column.The single reaction monitoring (m/z 116-57) shows specific for detection of 4-methyl- 2-hexaneamine [26].

Allegrand J developed Atmospheric pressure photoionization (APPI) mass spectrometry of guanine using tunable synchrotron VUV radiation. In this APPI source coupled with tunable VUV photon source. The ionization of guanine occurred by chemical reactions, as a function of photon energy [13].

Pascual-Teresa Sd developed LC-MS method for analysis of anthocyanins from from purple corn cob. The nine different types of anthocyanins are isolated and identified by LC-MS. In this LC coupled with diode array spectrometry and mass spectrometry and determined the anthocyanins components from purple corn cob by fragmentation patterns (MS spectra) [30].

Wang Y developed LC-MS method for analysing total resveratrol in Grape juice, Cranberry juice and in wine. Samples were analysed by using reverse phase HPLC with positive ion atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Resveratrol was detected in grape juice, cranberry juice and in wine, the concentration ranges from 1.56nmol/g, 1.07nmol/g and 8.63to 24.84 ^mol/L respectively [31].

Chang-Kee L describes the current developments in LC-MS for pharmaceutical analysis. In this techniques like electrospray, atmospheric chemical ionization and photo ionization and their interfaces are discussed. The LC-MS application in drug discovery, in vitro and in vivo drug metabolism, identification, characterization of impurities in pharmaceutical analysis has been briefly discussed [1].

Nishikawa M reported the determination of surfactants by using LC-MS in forensic toxicity. The analysis of anionic, cationic and non-ionic surfactants are done in both negative and positive mode i.e. anionic surfactants and positive surfactants are detected as M- ions in the negative mode and M+ ions in positive mode while non-ionic surfactants are in [M+H]+ ions or [M+NH4]+ ions in positive mode. The recovery range obtained for anionic, cationic and non-ionic surfactants are 65.8% to 124 % [32].

Hernando MD, determined the trace level of pharmaceutical residues in natural and treated water by using LC-MS. The samples like influent and effluent wastewaters, rivers and tap waters are analysed. The pharmaceuticals like Ibuprofen, Ketoprofen, and Diclofenac are determined at trace level by using solid -phase extraction (SPE) with liquid chromatography tandem mass spectrometry. The method detection limit and quantitation limit were 7.5-75 ng/L [33].

Souverain S developed the method for determination of protein precipitation for analysis of drug cocktail in plasma by using LC-ESI-MS. For protein precipitation (PP), Acetonitrile (ACN), perchloric acid (PA) and trichloroacetic acid (TCA) are used. The LC-ESI-MS method was developed for simultaneous analysis of six tested compounds in less than 6 minutes. Depending on the effective protein precipitation techniques to remove protein from human plasma and compatibility with LC-ESI-MS, ACN is used as PP technique in which recovery above 80% and CV up to 6% obtained [34].

Bogusz MJ , developed the LC-ESI-MS method for detection of synthetic adulterants in herbal remedies. Methanol is used for extraction of drugs and are separated by liquid chromatography with gradient mobile phase, Acetonitrile-10mM ammonium formate buffer pH 3.0. The limit of detection reported is 5 pg to 1 ng per injection of sample. The recoveries of spiked drugs obtained are from 63 to 100% [35].

Roach AGS, describes the application of LC-MS/MS method for analysis for Acrylamide in foods. Acrylamide is used as internal standard. The limit of quantitation [13 C3] is 10ppb (|ig/kg). The method is applied to variety of foods. The variability in analyte level in food type helps to decrease the acrylamide in foods [36].

Jangala H developed and validated the LC-MS method for simultaneous estimation of Amlodipine and Valsartan in human plasma. The chromatographic separation achieved by using isocratic method using mobile phase as Acetonitrile: 5mM ammonium formate solution (80:20 v/v), flow rate 0.8mL/ min. The quantitation was done using ESI in positive mode under multiple reactions monitoring (MRM) condition. Assay obtained in range 0.302-20.725ng/mL for Amlodipine and 6.062-18060.792 ng/mL) for Valsartan [37].


The LC-MS is a hyphenated technique used in combination with separation power of HPLC with detection power of Mass spectrometry. It is widely used in pharmaceutical, chemical, food, agrochemical industries, environmental and forensic applications. LC-MS is used for qualitative and quantitative determination of drug substances and biological samples. Also it is commonly used in drug research and quality control.

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