Iltaf Shah

Misleading measures in Vitamin D analysis: a novel LC-MS/MS assay to account for epimers and isobars

BackgroundRecently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars.MethodsA positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified.ResultsThe new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum.ConclusionsTo our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies.

Incongruence in Doping Related Attitudes, Beliefs and Opinions in the Context of Discordant Behavioural Data: In Which Measure Do We Trust?

Background Social psychology research on doping and outcome based evaluation of primary anti-doping prevention and intervention programmes have been dominated by self-reports. Having confidence in the validity and reliability of such data is vital. Methodology/Principal Findings The sample of 82 athletes from 30 sports (52.4% female, mean age: 21.48±2.86 years) was split into quasi-experimental groups based on i) self-admitted previous experience with prohibited performance enhancing drugs (PED) and ii) the presence of at least one prohibited PED in hair covering up to 6 months prior to data collection. Participants responded to questionnaires assessing a range of social cognitive determinants of doping via self-reports; and completed a modified version of the Brief Implicit Association Test (BIAT) assessing implicit attitudes to doping relative to the acceptable nutritional supplements (NS). Social projection regarding NS was used as control. PEDs were detected in hair samples from 10 athletes (12% prevalence), none of whom admitted doping use. This group of ‘deniers’ was characterised by a dissociation between explicit (verbal declarations) and implicit (BIAT) responding, while convergence was observed in the ‘clean’ athlete group. This dissociation, if replicated, may act as a cognitive marker of the denier group, with promising applications of the combined explicit-implicit cognitive protocol as a proxy in lieu of biochemical detection methods in social science research. Overall, discrepancies in the relationship between declared doping-related opinion and implicit doping attitudes were observed between the groups, with control measures remaining unaffected. Questionnaire responses showed a pattern consistent with self-reported doping use. Conclusions/Significance Following our preliminary work, this study provides further evidence that both self-reports on behaviour and social cognitive measures could be affected by some form of response bias. This can question the validity of self-reports, with reliability remaining unaffected. Triangulation of various assessment methods is recommended.

Method for simultaneous analysis of eight analogues of vitamin D using liquid chromatography tandem mass spectrometry

BackgroundDespite considerable global investigation over several decades, the roles of vitamin D in health and disease development remains convoluted. One recognised issue is the difficulty of accurately measuring the active forms of vitamin D. Advances made include some new methods addressing the potential interference by excluding epimers and isobars. However, there is no evidence that epimers are without function. Therefore, the aim of this study was to develop and validate, for the first time, a new assay to simultaneously measure levels of six forms of vitamin D along with two epimers. The assay was applied to multilevel certified reference material (CRM) and 25 pooled human sera samples, obtained from the Vitamin D External Quality Assessment Scheme (DEQAS), to demonstrate its efficiency.ResultsThe assay is capable of simultaneously measuring eight vitamin D analogues over the calibration ranges and LODs (in nmol/L) of: 1α25(OH)2D2 [0.015-1; 0.01], 1α25(OH)2D3 [0.1-100; 0.01], 25OHD3 [0.5-100, 0.025], 3-epi-25OHD3 [0.1-100, 0.05], 25OHD2 [0.5-100, 0.025], 3-epi-25OHD2 [0.1-100, 0.05], vitamin D3 [0.5-100, 0.05] and vitamin D2 [0.5-100, 0.05], using stanozolol-d3 as internal standard. Certified reference material and external quality control samples (DEQAS) were analysed to meet the standards outlined by National Institute of Standards and Technology (NIST). Validation steps included recovery and both precision and accuracy under inter- and intra-day variation limit of detection, and analysis of each analyte over a linear range. All validation parameters were in line with acceptable Food and Drug Administration (FDA) guidelines. All eight analogues were quantified with the 25OHD levels being commensurate with DEQAS data.ConclusionsThis report details the application of a new LC-MS/MS based assay for the efficient analysis of eight analogues of vitamin D over a range of samples, which is a significant advance over the existing methods. Simultaneous measure of eight vitamin D analogues does not compromise the analytical capability of the assay to quantify the commonly used biomarker (25OHD) for vitamin D status. The results demonstrate the feasibility of applying the assay in research and clinical practice that i) excludes misleading measures owing to epimers and isobars and ii) is able to quantify the excluded component to facilitate further in vivo investigation into the roles of ubiquitous epimers.

A theory-based online health behaviour intervention for new university students (U@Uni): results from a randomised controlled trial

BackgroundToo few young people engage in behaviours that reduce the risk of morbidity and premature mortality, such as eating healthily, being physically active, drinking sensibly and not smoking. This study sought to assess the efficacy and cost-effectiveness of a theory-based online health behaviour intervention (based on self-affirmation theory, the Theory of Planned Behaviour and implementation intentions) targeting these behaviours in new university students, in comparison to a measurement-only control.MethodsTwo-weeks before starting university all incoming undergraduates at the University of Sheffield were invited to take part in a study of new students’ health behaviour. A randomised controlled design, with a baseline questionnaire, and two follow-ups (1 and 6 months after starting university), was used to evaluate the intervention. Primary outcomes were measures of the four health behaviours targeted by the intervention at 6-month follow-up, i.e., portions of fruit and vegetables, metabolic equivalent of tasks (physical activity), units of alcohol, and smoking status.ResultsThe study recruited 1,445 students (intervention n = 736, control n = 709, 58% female, Mean age = 18.9 years), of whom 1,107 completed at least one follow-up (23% attrition). The intervention had a statistically significant effect on one primary outcome, smoking status at 6-month follow-up, with fewer smokers in the intervention arm (8.7%) than in the control arm (13.0%; Odds ratio = 1.92, p = .010). There were no significant intervention effects on the other primary outcomes (physical activity, alcohol or fruit and vegetable consumption) at 6-month follow-up.ConclusionsThe results of the RCT indicate that the online health behaviour intervention reduced smoking rates, but it had little effect on fruit and vegetable intake, physical activity or alcohol consumption, during the first six months at university. However, engagement with the intervention was low. Further research is needed before strong conclusions can be made regarding the likely effectiveness of the intervention to promote health lifestyle habits in new university students.Trial registrationCurrent Controlled Trials, ISRCTN67684181.

Recovery and Adaptation From Repeated Intermittent-Sprint Exercise

PURPOSE This investigation aimed to ascertain a detailed physiological profile of recovery from intermittent-sprint exercise of athletes familiar with the exercise and to investigate if athletes receive a protective effect on markers of exercise-induced muscle damage (EIMD), inflammation, and oxidative stress after a repeated exposure to an identical bout of intermittent-sprint exercise. METHODS Eight well-trained male team-sport athletes of National League or English University Premier Division standard (mean ± SD age 23 ± 3 y, VO2max 54.8 ± 4.6 mL ·kg-1 · min-1) completed the Loughborough Intermittent Shuttle Test (LIST) on 2 occasions, separated by 14 d. Maximal isometric voluntary contraction (MIVC), countermovement jump (CMJ), creatine kinase (CK), C-reactive protein (CRP), interleukin-6 (IL-6), F2-isoprostanes, and muscle soreness (DOMS) were measured before and up to 72 h after the initial and repeated LISTs. RESULTS MIVC, CMJ, CK, IL-6, and DOMS all showed main effects for time (P < .05) after the LIST, indicating that EIMD was present. DOMS peaked at 24 h after LIST 1 (110 ± 53 mm), was attenuated after LIST 2 (56 ± 39 mm), and was the only dependent variable to demonstrate a reduction in the second bout (P = .008). All other markers indicated that EIMD did not differ between bouts. CONCLUSION Well-trained games players experienced EIMD after exposure to both exercise tests, despite being accustomed to the exercise type. This suggests that well-trained athletes receive a very limited protective effect from the first bout.

Antioxidant Enzymatic Activities in Alzheimer's Disease: The Relationship to Acetylcholinesterase Inhibitors

The mode of action of acetylcholinesterase inhibitors (AChEIs) in Alzheimers disease (AD) is mainly by potentiating neuronal transmission. Animal studies have also consistently described a role for AChEIs in enhancement of antioxidants and attenuation of oxidative stress. The influence of AChEIs on blood antioxidants in AD patients has not been established before. Furthermore, AChEI treatment, or lack of it, may have contributed to the inconsistent antioxidant data reported by other studies so far. Here we sought to investigate the potential modulation effect of AChEIs on blood antioxidants in AD patients. Catalase (CAT) and glutathione reductase (GR) activities were analyzed in 25 drug naïve patients (Group A), 43 patients receiving AChEIs (Group B) and 34 cognitively unimpaired controls (Group C). A statistically significant difference for CAT and GR was observed between the two AD groups (A and B) when compared to the control group C (KW-H = 36.530, p < 0.001; post hoc tests p < 0.001 and KW-H = 37.814, p < 0.001; post hoc tests p < 0.001, respectively). In contrast, CAT and GR activities did not differ significantly between the two AD groups, and were not influenced by AChEI treatment. Hence, these results do not replicate the extensively reported data from animal studies and question whether AChEI efficacy in AD is mediated by processes beyond neuron to neuron enhancement of transmission. Studies assessing a wider range of oxidative/inflammatory markers taking into account type, dosage, and treatment duration of the various acetylcholinesterase inhibitors are now needed.

Exploring the Role of Vitamin D in Type 1 Diabetes, Rheumatoid Arthritis, and Alzheimer Disease: New Insights From Accurate Analysis of 10 Forms

CONTEXT AND OBJECTIVE A comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to quantify 10 forms of vitamin D in sera from healthy adults and patients suffering from rheumatoid arthritis (RA), type 1 diabetes (T1-D), and Alzheimer disease (AD). DESIGN The rapid assay, validated according to US Food and Drug Administration guidelines with Chromsystems and DEQAS samples, was applied to 36 nonhealthy sera samples (41.7% male, age range of 14-95, mean = 54.00 ± 21.98 years), consisting of individuals with RA, T1-D, and AD (n = 12 each) and was compared to samples from 32 healthy individuals (50% male, age range of 19-90, mean = 58.83 ± 22.93 years). RESULTS The key findings are (1) the 23R,25-dihydroxyvitamin D3 form was quantified for the first time (healthy = 0.427 ± 0.633 nmol/L; combined disease = 0.395 ± 0.483 nmol/L), (2) the 3-epi-25-hydroxyvitamin D3 metabolite was found in all groups with significantly higher concentration in the diseased samples [healthy = 6.093 ± 6.711 nmol/L; combined disease = 22.433 ± 13.535 nmol/L, t(52.5) = -6.411; P < .001], (3) a significant difference was found for the active form (1α-25-dihydroxyvitamin D3) between health (0.027 ± 0.035 nmol/L) and disease (0.433 ± 0.870 nmol/L) [t(35.1) = -2.797, P = 0.008], and (4) there was no significant correlation between the total circulating and total active forms in either the disease or healthy group (r = -0.180 and -0.274, respectively, with no difference between the correlation coefficients, z = -0.389, P = .697). Receiver operating characteristic curve analysis showed good sensitivity and specificity for using the 3-epi-25-hydroxyvitamin D concentration to predict disease status (area under the curve = 0.880, P < .001). Discriminant function analysis using concentrations of 23R,25-dihydroxyvitamin D3, 25-hydroxyvitamin D2, and 3-epi-25-hydroxyvitamin D classified 94.4% (91.7% in cross-validation) of the cases correctly. CONCLUSIONS This study reveals significant differences between health and disease with epimers having the potential to relate to disease. The potential implications of the information gleaned from measuring all forms warrant application of more comprehensive assays for future clinical studies investigating the link between vitamin D and health.

Russian roulette with unlicensed fat-burner drug 2,4-dinitrophenol (DNP): evidence from a multidisciplinary study of the internet, bodybuilding supplements and DNP users.

Background2,4-Dinitrophenol (DNP) poses serious health-risks to humans. The aims of this three-stage multidisciplinary project were, for the first time, to assess the risks to the general public from fraudulent sale of or adulteration/contamination with DNP; and to investigate motives, reasons and risk-management among DNP-user bodybuilders and avid exercisers.MethodsUsing multiple search-engines and guidance for Internet research, online retailers and bodybuilding forums/blogs were systematically explored for availability of DNP, advice offered on DNP use and user profiles. Ninety-eight pre-workout and weight-loss supplements were purchased and analysed for DNP using liquid-chromatography-mass-spectrometry. Psychosocial variables were captured in an international sample of 35 DNP users (26.06 ± 6.10 years, 94.3 % male) with an anonymous, semi-qualitative self-reported survey.ResultsAlthough an industrial chemical, evidence from the Internet showed that DNP is sold ‘as is’, in capsules or tablets to suit human consumption, and is used ‘uncut’. Analytical results confirmed that DNP is not on the supplement market disguised under fictitious supplement names, but infrequently was present as contaminant in some supplements (14/98) at low concentration (<100mcg/kg). Users make conscious and ‘informed’ decisions about DNP; are well-prepared for the side-effects and show nonchalant attitude toward self-experimentation with DNP. Steps are often taken to ensure that DNP is genuine. Personal experience with performance- and appearance enhancing substances appears to be a gateway to DNP. Advice on DNP and experiences are shared online. The significant discrepancy between the normative perception and the actual visibility suggests that DNP use is-contrary to the Internet accounts-a highly concealed and lonesome activity in real life. Positive experiences with the expected weight-loss prevail over the negative experiences from side effects (all but two users considered using DNP again) and help with using DNP safely is considered preferable over scare-tactics.ConclusionLegislation banning DNP sale for human consumption protects the general public but DNP is sold ‘as is’ and used ‘uncut’ by determined users who are not dissuaded from experimenting with DNP based on health threats. Further research with stakeholders’ active participation is imperative for targeted, proactive public health policies and harm-reduction measures for DNP, and other illicit supplements.

Simultaneous analysis of antiretroviral drugs abacavir and tenofovir in human hair by liquid chromatography–tandem mass spectrometry

A sensitive and reproducible method has been developed and validated for the simultaneous quantification of the key antiretroviral drugs abacavir and tenofovir in hair using LC-MS/MS. The method was validated according to the US Food and Drug Administration (FDA) guidelines for the parameters: specificity, stability, limits of detection (LOD), limits of quantification (LOQ), linearity, accuracy, precision and recovery. Hair samples (50mg) were decontaminated and subjected to methanolic extraction, where 1 ml methanol was added along with the internal standard abacavir-d4 at a final concentration of 0.15 ng/mg hair. After 16 h, the drugs were recovered by liquid-liquid extraction using ammonium acetate buffer and a mixture of methyl tert-butyl ether:ethyl acetate (1:1). The samples were reconstituted with 200 μl acetonitrile:water (1:1) prior to injection for LC-MS/MS. The LOD and LOQ values were 0.06 and 0.12 ng/mg (drug/hair) for both drugs. Calibration curves were linear in the concentration range of 0.12-4.0 ng/mg of drug/hair with regression coefficient (r(2)) value of 0.999 for both drugs. The data for accuracy, precision and recovery were within the FDA limits. The concentrations of the drugs in the hair samples ranged from 0.12 ng/mg to 4.48 ng/mg and 0.32 ng/mg to 1.67 ng/mg for tenofovir and abacavir, respectively. This is the first full report of a method for the simultaneous determination of these two key antiretroviral drugs in hair. The newly developed method is useful for future routine analysis of tenofovir and abacavir in human hair and could be used in therapeutic drug monitoring and adherence to medicines studies, which would be helpful in decision making regarding treatment change in combination anti-retroviral therapies.

LC-MS/MS-Based Assay for Free and Deconjugated Testosterone and Epitestosterone in Rat Urine and Serum

Testosterone and epitestosterone are mainly excreted as glucuronides. The aim of this study was to develop and validate a method using liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyse testosterone and epitestosterone in rat serum and urine to assist in vivo studies on steroid metabolism. The method was developed by spiking charcoal stripped rat plasma and urine with the analytes. The developed method was then applied to serum (n=6) and urine samples (n=6) from young male brown Norway rats to determine testosterone and epitestosterone concentrations. The assay showed linearity within quantification range coefficient (r2) values above 0.991. Optimum conditions were determined for the deconjugation of glucuronidated testosterone and epitestosterone along with the internal standard stanozolol D3. Accuracy, precision and extraction recovery for both compounds was satisfactory in both matrices. The method was capable of quantifying 0.250 ng/mL concentrations of testosterone and epitestosterone in 100 μL of serum and urine. The average concentrations of free and deconjugated testosterone and epitestosterone found in the rat samples were: urine–201.68 ± 90.16 ng/mL and 85.37 ± 21.20 ng/mL; serum– 363.40 ± 11.615 ng/mL and 1.75 ± 0.118 ng/mL, respectively. This method is sensitive, specific and reproducible for the determination of free and deconjugated testosterone and epitestosterone in rat serum and urine. The method can be used for in vivo analysis for further investigations of testosterone and epitestosterone concentrations in studies monitoring endocrine dysfunctions and doping.