Development and validation of a dried blood spots assay for metabolic profiling of ginsenosides using ultra-high performance liquid chromatography-mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C.A. Meyer., a famous and valuable traditional Chinese medicine with thousand years of history for its healthcare and therapeutic effects. It is necessary and meaningful to study the pharmacokinetic behavior of ginsenosides in vivo as they are the most active components. Dried blood spots (DBS) are a mature and advanced blood collection method with meet the needs for the measurement of numerous analytes. AIM OF THE STUDY: This study aimed to explore the feasibility on DBS in the metabolic profile analysis of complex herbal products. MATERIALS AND METHODS: An ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of ginsenosides. The preparation of DBS samples was conducted by spiking the whole blood with analytes to obtain 20 µL of blood spots on Whatman 903 collection card. A punched dish of 10 mm in diameter was extracted with 70 % methanol aqueous solution, digoxin was used as an internal standard. Target compounds were separated on a Waters T3 column (2.1 × 100 mm, 1.8 µm) with acetonitrile and water (0.1 % formic acid) at a flow rate of 0.4 mL/min. RESULTS: The various ginsenosides showed good linearity in the range of 1-2000 ng/mL. The extraction recoveries and matrix effects of the target analytes were above 82.2%. The intra- and inter-batch accuracy and precision were within the limits of ≤15% for all tested concentrations. Moreover, the collected dried blood spot samples could be stably stored at room temperature for 14 days and 4 °C for 1 month without being affected. And it is delightful that the DBS-based analysis is compatible or even superior to the conventional protein precipitation in terms of sensitivity, linearity, and stability. In particular, the target analytes are stable in the DBS sampling under normal storing condition and the sensitivity for some trace metabolites of ginsenosides, such as 20(S)-Rg3, 20(R)-Rg3, F1, Rk1, Rg5, etc. increases 3-4 folds as evaluated by LLOQ. CONCLUSIONS: The established method was successfully applied to pharmacokinetic studies of ginseng extract in mice, this suggests a more feasible strategy for pharmacokinetic study of traditional and natural medicines both in animal tests and clinical trials.

The reciprocal associations between social deficits, social engagement, and inflammation: Longitudinal evidence comparing venous blood samples and dried blood spots and mapping the modifying role of phenotypic and genotypic depression.

BACKGROUND: Social psychoneuroimmunology suggests an interplay between social deficits (loneliness and isolation) and chronic inflammation, but the direction of these relationships remains unclear. We estimated the reciprocal associations of social deficits and social engagement with levels of C-reactive protein (CRP), compared the consistency of the findings depending on the biological sampling method used, and examined the modifying role of phenotypic and genotypic depression. METHODS: We used longitudinal nationally representative data from the US (Health and Retirement Study, 3 waves, 2006-16) and England (English Longitudinal Study of Ageing, 4 waves, 2004-18). Loneliness, social isolation, and social engagement were self-reported. CRP was measured using dried blood spots (US) and venous blood samples (England). Cross-lagged panel models were fitted and tested interactions with phenotypic depression (above-threshold depressive symptom scores) and genotypic depression (polygenic score for major depressive disorder). RESULTS: We included 15,066 participants (mean age = 66.1 years, SD = 9.8) in the US and 10,290 (66.9 years, SD = 10.5) in England. We found reciprocal associations between loneliness and CRP using dried blood spots and venous blood samples. Higher CRP predicted higher subsequent loneliness and higher loneliness predicted elevated CRP. Both phenotypic and genotypic depression modified this reciprocal association. There were also reciprocal associations for social engagement in venous blood samples: higher CRP predicted lower social engagement and greater social engagement predicted lower subsequent CRP. Associations between social isolation and CRP were inconsistent and unidirectional. CONCLUSIONS: Loneliness may increase chronic inflammation, whereas social engagement may reduce inflammation. As these relationships were reciprocal, there may be a loop between inflammation, loneliness, and social engagement. This loop was stronger in those with depression or at high genetic risk for major depressive disorder. This relationship for loneliness was present in both blood sampling methods despite contrasting methods of CRP measurement, indicating that the finding is not attributable to measurement bias in biomarkers.

Field evaluation of HIV-1 viral load monitoring in adults and children receiving antiretroviral treatment in Nigeria by dried blood spot testing with RealTime HIV-1 on m2000.

In resource-limited settings, use of dried blood spots (DBS) could be a pragmatic alternative to plasma for VL monitoring in people living with HIV (PLWH). We compared results from DBS to standard plasma VL testing under field conditions in patients receiving antiretroviral therapy (ART). DBS cards were prepared from venous blood (V-DBS), finger-pricks using micro-capillary tubes (M-DBS), and direct spotting (D-DBS). DBS and matched EDTA plasma were tested on the Abbott m2000 platform using the appropriate RealTime HIV-1 quantitative CE protocol. Matched plasma samples were also tested on the Roche COBAS Ampliprep/COBAS TaqMan version 2.0. Diagnostic accuracy indicators (sensitivity, specificity, misclassification rate, and kappa coefficient) for viral failure (VF) based on different VL threshold levels and agreement of absolute VL were calculated. A total of 669 participants provided 2676 samples. V-DBS had a peak sensitivity for VF of 89.1 % [95 % CI: 85.5-92.7] at the 1000 copies/mL threshold and a peak specificity of 97.4 % [95 % CI: 95.9-99.0] at the 5000 copies/mL threshold. The lowest proportion of upward misclassification (patients classified with VF who actually had viral suppression) for V-DBS was 3.1 % [95 % CI: 1.4-4.8] at the 5000 copies/mL threshold, whereas the lowest proportion of downward misclassification (patients classified as undetectable who actually had VF) was 10.9 % [95 % CI: 7.2-14.5] at the 1000 copies/mL threshold. Abbott RealTime HIV-1 VL results from all 3 DBS types for adults and children showed strong correlation with the gold standard plasma-based assay. DBS could be useful for monitoring VL in resource limited settings such as Nigeria.

Volumetric absorptive microsampling (VAMS®) in therapeutic protein quantification by LC-MS/MS: Investigation of anticoagulant impact on assay performance and recommendations for best practices in method development.

Microsampling techniques have been employed as an alternative to traditional serum/plasma sampling because of their inherently proven and desirable advantages across the pharmaceutical industry. These include reduced animal usage in pre-clinical studies, as well as, permitting the collection of samples that would otherwise be inaccessible in clinical studies. The application of volumetric absorptive microsampling (VAMS®) technology, a second-generation dried microsampling method, coupled with LC-MS, has been extensively explored for small molecule drugs at various drug development stages. However, the potential of using VAMS technology and LC-MS analysis for biological therapeutic development has yet to be well-established. In this work, we describe the method development, validation, and a proof-of-concept non-human primate study of a LC-MS/MS method for VAMS utilized to obtain pharmacokinetic (PK) data for a therapeutic monoclonal antibody. A good correlation between VAMS data and data from conventional serum samples was established in rhesus monkeys and indicated the possibility of using of this novel sampling technology in clinical studies. However, during the initial clinical study, a significant difference in internal standard (IS) response between the patient fingerstick samples and the standard/QC samples was observed, which posed a question on the accuracy of the clinical results. A comprehensive investigation confirmed that the EDTA anticoagulant used in the standard/QC samples was the root cause of the observed anomalous IS responses. Special considerations and corresponding best practices during method development and validation are proposed to ensure early detection of potential issues and appropriate implementation of VAMS technology in clinical studies in the future.

Evaluation of dried blood spots as an alternative matrix for therapeutic drug monitoring of abiraterone and delta(4)-abiraterone in prostate cancer patients.

Therapeutic drug monitoring (TDM) approaches may benefit patients treated with abiraterone acetate (AA) as drug efficacy is imprecise and important pharmacokinetic variability is known. Current methods based on the analysis of plasma present the disadvantage of the fast degradation of the analytes in the liquid sample. Dried blood spots (DBS) consist of a minimally invasive and unexplored sampling strategy to monitor the levels of abiraterone (ABI) and delta(4)-abiraterone (D4A) in patients. This study presents the development and validation of a precise and accurate method to monitor ABI and D4A in DBS samples by UPLC-MS/MS. Bioanalytical method validation was carried out according to current guidelines, evaluating the impact of DBS-specific parameters such as hematocrit and spot volume on accuracy. Based on the analysis of quality control samples prepared at low, medium and high concentrations, the method was precise with CV ≤ 6.97 % and 10.26 % for ABI and D4A, respectively. The method was also highly accurate, between 93.6-106.8 % for ABI and 96.0-108.5 % for D4A. The DBS method is compatible with the analysis of samples of unknown volume and hematocrit range of the studied population. In addition, ABI and D4A were stable for 7 days in DBS at room temperature, which is feasible for sample transportation in postal service and analysis in the laboratory. Method application to 16 clinical samples revealed good correlation between measured plasma concentrations and estimated plasma concentrations for ABI (r = 0.884, P < 0.05) and D4A (r = 0.920, P < 0.05). Passing-Bablok regression analysis and Bland-Altmann plots indicated correlation between the results obtained from DBS and plasma, with a slight overestimation of the concentrations of ABI in DBS, which could be related to the small study cohort. Therefore, the results of this first work indicate that DBS consist of a promising alternative sampling strategy in TDM studies of AA.

Postprandial Dried Blood Spot-Based Nutritional Metabolomic Analysis Discriminates a High-Fat, High-Protein Meat-Based Diet from a High Carbohydrate Vegan Diet: A Randomized Controlled Crossover Trial.

BACKGROUND: Due to the challenges associated with accurate monitoring of dietary intake in humans, nutritional metabolomics (including food intake biomarkers) analysis as a complementary tool to traditional dietary assessment methods has been explored. Food intake biomarker assessment using postprandial dried blood spot (DBS) collection can be a convenient and accurate means of monitoring dietary intake vs 24-hour urine collection. OBJECTIVE: The objective of this study was to use nutritional metabolomics analysis to differentiate a high-fat, high-protein meat (HFPM) diet from a high-carbohydrate vegan (HCV) diet in postprandial DBS and 24-hour urine. DESIGN: This was a randomized controlled crossover feeding trial. PARTICIPANTS/SETTING: Participants were healthy young adult volunteers (n = 8) in California. The study was completed in August 2019. INTERVENTION: The standardized isocaloric diet interventions included an HFPM and an HCV diet. Participants attended 2 intervention days, separated by a 2-week washout. MAIN OUTCOME MEASURES: During each intervention day, a finger-prick blood sample was collected in the fasting state, 3 hours post breakfast, and 3 hours post lunch. Participants also collected their urine for 24 hours. DBS and urine samples were analyzed by ultra-performance liquid chromatography mass spectrometry to identify potential food intake biomarkers. STATISTICAL ANALYSES PERFORMED: Principal component analysis for discriminatory analysis and univariate analysis using paired t tests were performed. RESULTS: Principal component analysis found no discrimination of baseline DBS samples. In both the postprandial DBS and 24-hour urine, post-HFPM consumption had higher (P < 0.05) levels of acylcarnitines, creatine, and cis-trans hydroxyproline, and the HCV diet was associated with elevated sorbitol (P < 0.05). The HFPM diet had higher concentrations of triacylglycerols with fewer than 54 total carbons in DBS, and 24-hour urine had higher nucleoside mono- and di-phosphates (P < 0.05). CONCLUSIONS: Nutritional metabolomics profiles of postprandial DBS and 24-hour urine collections were capable of differentiating the HFPM and HCV diets. The potential use of postprandial DBS-based metabolomic analysis deserves further investigation for dietary intake monitoring.

Failure of national antenatal vitamin D supplementation programme puts dark skinned infants at highest risk: A newborn bloodspot screening study.

OBJECTIVES: To determine the prevalence of vitamin D deficiency on dried blood spots (DBS) obtained at newborn blood spot screening (NBS) and thereby test the efficacy of the UK national antenatal supplementation programme in an increasingly ethnically diverse English population. To evaluate the seasonal and ethnic variation in neonatal plasma 25 hydoxyvitamin D (25OHD) and its determinants. DESIGN: Three thousand random DBS samples received at a single regional newborn screening laboratory (52° N) over two one-week periods, one in winter (February 2019) and one in summer (August 2019), were collected. Data was collected from NBS cards on birth weight, gestational age, maternal age, ethnicity, and post code which was replaced with index of multiple deprivation (IMD). 25OHD concentrations were measured on 6 mm sub-punch from DBS using quantitative liquid chromatography tandem mass spectrometry adjusted to equivalent plasma values. 25OHD variation with season was assessed using Mann-Whitney U test and ethnic groups compared using Kruskal-Wallis test. Linear regression was used to assess the determinants of 25OHD concentrations. RESULTS: 25OHD measurements were available in 2999 (1580 males) subjects [1499 winter-born and 1500 summer-born]. The majority were white British (59.1%) and born at term (mean ± SD gestational age of 38.8 ± 1.8 weeks) with a mean (±SD) birth weight of 3306 (±565) grams. The overall prevalence of vitamin D deficiency [25OHD<30 nmol/L (12 µg/L)] was 35.7% (n = 1070) and insufficiency [30-50 nmol/L (12-20 µg/L)] 33.7% (n = 1010). The median (IQR) 25OHD concentration was significantly lower in the winter-born compared to summer-born [29.1 (19.8, 40.6) vs 49.2 (34.3, 64.8) nmol/L respectively; p < 0.001]. Across both seasons, when compared to white British babies (41.6 nmol/L), the median 25OHD concentrations were significantly lower in babies of black (30.3 nmol/L; p < 0.001), Asian (31.3 nmol/L; p < 0.001), any other mixed (32.9 nmol/L; p < 0.001), mixed white and black (33.7 nmol/L; p < 0.05) and any other white (37.7 nmol/L; p < 0.05) ethnicity. The proportion of deficiency was also higher in babies of Asian (48%), black (47%) and mixed ethnicity (38-44%) compared to any other white (34%) or white British (30%) ethnicity. Season of birth, ethnicity, gestation and maternal age accounted for almost 24% of the variation in 25OHD concentrations. CONCLUSION: The current UK antenatal supplementation programme fails to protect newborns from vitamin D deficiency, especially those from minority ethnic groups who are at high risk of vitamin D deficiency. Nearly 70% of all newborns and 85% of winter-borns had 25OHD concentrations below 50 nmol/L (20 µg/L). Almost 50% of babies of Black or Asian origin were deficient at birth, which explains their high risk of hypocalcaemic complications and rickets if left unsupplemented. Our findings call for an immediate review of the delivery of antenatal and infant vitamin D supplementation programmes and implementation of food fortification in the long term.

DBS Assay with LC-MS/MS for the Determination of Idelalisib, A Selective PI3K-δ Inhibitor in Mice Blood and Its Application to a Pharmacokinetic Study.

Idelalisib is a selective and second-generation PI3K-δ inhibitor, approved for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this paper, we present a fully validated dried blood spot (DBS) method for the quantitation of idelalisib from mice blood using an LC-MS/MS, which was operated under multiple reaction monitoring mode. To the punched DBS discs, acidified methanol enriched with internal standard (IS; larotrectinib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of idelalisib and the IS was achieved on an Atlantis dC18 column using a mixture of 10 mM ammonium formate:acetonitrile (25:75, v/v). The flow-rate and injection volume were 0.80 mL/min and 2.0 µL, respectively. Idelalisib and the IS were eluted at ~0.98 and 0.93 min, respectively and the total run time was 2.00 min. Idelalisib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 416.1→176.1 and 429.1→342.1, respectively was used for the quantitation. The calibration range was 1.01-4 797 ng/mL. No matrix effect and carry over were observed. Haematocrit did not influence DBS idelalisib concentrations. All the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mice pharmacokinetic study.

Remote testing of vitamin D levels across the UK MS population-A case control study.

OBJECTIVE: The association between vitamin D deficiency and multiple sclerosis (MS) is well described. We set out to use remote sampling to ascertain vitamin D status and vitamin D supplementation in a cross-sectional study of people with MS across the UK. METHODS: People with MS and matched controls were recruited from across the UK. 1768 people with MS enrolled in the study; remote sampling kits were distributed to a subgroup. Dried blood spots (DBS) were used to assess serum 25(OH)D in people with MS and controls. RESULTS: 1768 MS participants completed the questionnaire; 388 MS participants and 309 controls provided biological samples. Serum 25(OH)D was higher in MS than controls (median 71nmol/L vs 49nmol/L). A higher proportion of MS participants than controls supplemented (72% vs 26%, p<0.001); people with MS supplemented at higher vD doses than controls (median 1600 vs 600 IU/day, p<0.001). People with MS who did not supplement had lower serum 25(OH)D levels than non-supplementing controls (median 38 nmol/L vs 44 nmol/L). Participants engaged well with remote sampling. CONCLUSIONS: The UK MS population have higher serum 25(OH)D than controls, mainly as a result of vitamin D supplementation. Remote sampling is a feasible way of carrying out large studies.

Diagnostic Challenges Using a 2-Tier Strategy for Methylmalonic Acidurias: Data from 1.2 Million Dried Blood Spots.

BACKGROUND: The detection of methylmalonic acid (MMA) by second-tier analysis has been shown to reduce the number of false positives in newborn screening (NBS) for genetically determined methylmalonic acidurias (MMAuria). In addition to genetic conditions, MMA is an indicator of vitamin B12 status, thus applicable to detect maternal vitamin B12 deficiency in the newborns screened. METHODS: Biochemical and clinical follow-up data of a 7.5-year pilot study with 1.2 million newborns screened were reviewed. RESULTS: Among 1,195,850 NBS samples, 3,595 (0.3%) fulfilled criteria for second-tier analysis of MMA. In 37 (0.003%; 1/32,000) samples, elevated concentrations of MMA were detected, resulting in diagnostic workup at a metabolic center in 21 newborns. In 6 infants (1/199,000), genetic conditions were established, 1 infant with cobalamin C deficiency (CblC) showed only a moderate elevation of MMA. The remaining 15 newborns (1/79,000) displayed significantly lower concentrations of MMA and were evaluated for maternal vitamin B12 deficiency. In 9 mothers, vitamin B12 deficiency was verified, and 6 showed no indication for vitamin B12 deficiency. Treatment with vitamin B12 normalized biochemical parameters in all 15 infants. CONCLUSIONS: Applying a 2-tier strategy measuring MMA in NBS identified genetic conditions of MMAuria. It was possible to separate severe, early-onset phenotypes from maternal vitamin B12 deficiency. However, the detection of CblC deficiency with mildly elevated MMA interferes with impaired vitamin B12 status of unknown relevance and thus burdens possibly healthy newborns. Regarding maternal vitamin B12 deficiency, testing and supplementing mothers-to-be is preferable. This might decrease straining follow-up of newborns and improve quality and overall perception of NBS.

Measuring thiamine status in dried blood spots.

BACKGROUND: A reliable and robust method with minimum sample collection requirement for thiamine assay is needed in clinical and research settings. METHODS: A simple and robust assay for three vitamers (thiamine, Th; thiamine monophosphate, TMP; and thiamine diphosphate, TDP) using a 6.35-mm dried blood spot (DBS) disc was developed, validated and applied. RESULTS: We were able to quantify accurately thiamine status covering all major vitamers Th, TMP and TDP with acceptable recovery (90%-114%), limit of quantification (TDP: 3.0 nM, TMP and Th: 1.5 nM), linearity (TDP: LOQ 400 nM, TMP and Th: LOQ 50 nM, all R2 > 0.99), imprecision (coefficient variation < 4.3% for TDP, <10.0% for TMP and < 12.6% for Th) and stability at -20 °C for up to 42 days. By recruiting 20 healthy participants, we cross compared finger capillary DBS with venous whole blood and venous blood pre-spotted on filter papers. The results demonstrated minimum bias between methods. A preliminary dosing study showed the method had excellent sensitivity after a single dose of supplemental thiamine. CONCLUSIONS: We have developed and clinically validated a simple, robust, accurate and sensitive assay for the analysis of thiamine status in DBS, suitable for large-scale population studies.

Circulating Insulin-Like Growth Factor-1 Is Positively Associated with Growth and Cognition in 6- to 9-Year-Old Schoolchildren from Ghana.

BACKGROUND: Milk intake stimulates linear growth and improves cognition in children from low-income countries. These effects may be mediated through insulin-like growth factor-1 (IGF-1). OBJECTIVE: The objective was to assess the effect of milk supplement on circulating IGF-1 and to assess IGF-1 as a correlate of growth and cognition in children. METHODS: Secondary data on blood spot IGF-1 from a randomized, double-blind, controlled trial in 6-9-y-old children from rural Ghana were analyzed. Intervention groups received porridge with non-energy-balanced supplements: 8.8 g milk protein/d, 100 kcal/d (Milk8); 4.4 g milk and 4.4 g rice protein/d, 100 kcal/d (Milk/rice); 4.4 g milk protein/d, 48 kcal/d (Milk4); or a control (no protein, 10 kcal/d). IGF-1, length, body composition, and Cambridge Neuropsychological Test Automated Battery (CANTAB) were measured at 3.5 or 8.5 mo. Linear regressions were used to assess the effect of milk interventions on IGF-1 and IGF-1 as a correlate of growth and cognition. RESULTS: The increase in IGF-1 was 15.3 (95% CI: 3.3, 27.3) ng/mL higher in children receiving Milk8 compared with the control. The IGF-1 increases in the isonitrogenous, isoenergetic Milk/rice or the Milk4 groups were not different from the control (P ≥ 0.49). The increase in IGF-1 was associated with improvements in 4 out of 5 CANTAB domains. The strongest associations included reductions in "mean correct latency" from Pattern Recognition Memory and "pre-extradimensional (pre-ED) shift errors" from Intra/Extradimensional Set Shift (P ≤ 0.005). In addition, change in IGF-1 was positively associated with changes in height, weight, and fat-free mass (P ≤ 0.001). CONCLUSIONS: Intake of skimmed milk powder corresponding to one, but not half a glass of milk on school days stimulates IGF-1 in 6-9-y-old Ghanian children. IGF-1 seems to mediate the effect of milk intake on growth and cognition. The association between IGF-1 and cognition in relation to milk intake is novel and opens possibilities for dietary interventions to improve cognition.

Iodine deficiency in pregnant women in Sweden: a national cross-sectional study.

PURPOSE: Voluntary salt iodization at 50 mg/kg salt ensures adequate iodine nutrition in Swedish school-aged children, but iodine status in pregnant women is uncertain. METHODS: We conducted a cross-sectional national study of 743 pregnant women, at median gestational age of 23 weeks (IQR 9, 38), recruited from maternal health care centers. We measured: urinary iodine concentration (UIC) and urinary creatinine concentration in spot urine samples; thyroglobulin (Tg), thyroid-stimulating hormone (TSH), and total thyroxine (tT4) on dried blood spots (DBS); and thyreoperoxidase antibodies in serum samples. Data on dietary supplement use were obtained, and women were classified as supplement users (consuming multivitamins containing ≥ 150 µg iodine/day) and non-supplement users (no supplements or < 150 µg iodine/day from supplements). RESULTS: Overall median UIC [bootstrapped 95% confidence interval (CI)] was 101 µg/L (95, 108; n = 737): 149 µg/L (132, 164) in supplement users (n = 253) and 85 µg/L (79, 92) in non-supplement users (n = 440) (p < 0.001). Overall geometric mean DBS-Tg (95% CI) was 22.1 µg/L (20.8, 23.5; n = 675) and the prevalence of elevated DBS-Tg was 19%. DBS-Tg was lower in supplement users (n = 229) than in non-supplement users (n = 405) (19.1 vs 24.4 µg/L, p < 0.001). DBS-TSH, DBS-tT4, and S-TPOab positivity did not differ between the two groups. CONCLUSIONS: Pregnant women in Sweden have inadequate iodine nutrition. Women not taking iodine supplements containing ≥ 150 µg iodine/day are affected by mild iodine deficiency and are at higher risk for increased thyroid activity, while maintaining euthyroidism. Iodine intake should be improved in women both before and after conception by promotion of iodized salt instead of non-iodized salt. We urge regular monitoring of iodine status in the general Swedish population, as well as in risk groups.

New Advances in Amino Acid Profiling in Biological Samples by Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) offers a high efficiency microseparation platform for amino acid profiling when analyzing volume-restricted biological samples, such as a dried blood spot punch. Direct analysis of amino acids and their analogs is routinely achieved using strongly acidic buffer conditions under positive-ion mode detection with a coaxial sheath liquid interface for electrospray ionization (ESI). New advances in online sample preconcentration, pre-column chemical derivatization, and/or low flow/sheathless CE-MS interface designs can further improve sensitivity while allowing for resolution of amino acid stereoisomers and labile aminothiols with low nanomolar detection limits. Additionally, multiplexed separations in CE-MS based on serial injection of seven or more samples within a single run greatly boosts sample throughput (<2-3 min/sample) without added infrastructure costs while allowing for stringent quality control and signal batch correction. Accurate prediction of the electromigration behavior of amino acids and their analogs offers a convenient approach for structural elucidation that is complementary to high-resolution MS and MS/MS. Simultaneous analysis of amino acids together with other classes of ionic metabolites by CE-MS allows for comprehensive metabolomic screening as required for new advances in clinical medicine, nutritional sciences, and population health.

Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC-fluorimetry.

Thiamine deficiency, if detected early in infancy, can be treated with thiamine supplementation and can prevent seizures, other disabilities and death. The dried blood spot (DBS) sampling technique is an attractive sample collection technique for infants. The present study reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from DBS. The method utilizes full-spot analysis of a volumetrically deposited 40 µl DBS. The analyte was extracted from the DBS using 50% methanol and then derivatized using potassium ferricyanide to thiochrome. Separation was achieved with the help of an Inertsil ODS C18 column (5.0 µm, 250 × 4.6 mm) using 150 mm phosphate buffer pH 7-acetonitrile (90:10, % v/v) as the mobile phase. The use of a fluorimetric detector gave a good response to the thiochrome derivative offering good sensitivity for the method. The excitation and emission wavelengths were 367 and 435 nm, respectively. The limit of detection and lower limit of quantification were 5 and 10 ng/ml, respectively. Linearity was demonstrated from 10 to 1000 ng/ml, and precision (CV) was <12.08%, at all tested quality control levels. The method accuracy was 89.34-118.89% with recoveries >80%. Bland-Altman analysis of DBS sampling vs. whole blood demonstrated a mean bias of only 1.16 ng/ml, with a majority of the 60 investigated patient samples lying within 7.2% of the corresponding concentration measured in blood, thereby meeting the clinical desirable biological specification criterion and showing that the two methods are comparable.

Microsampling for quantitative bioanalysis, an industry update: output from an AAPS/EBF survey.

There is continuing interest in the development and application of various microsampling technologies for drug development. The AAPS bioanalytical community microsampling subgroup and the European Bioanalysis Forum conducted a survey of their members (39 individual organizations). This gives a snapshot of current practices and demonstrates that implementation of microsampling approaches is becoming increasingly commonplace, but not universal. Greater adoption was observed for nonclinical studies, particularly nonregulatory. A number of respondents reported that they have included microsampling data in regulatory submissions. Another important observation was that where microsampling is employed for clinical studies, dried blood approaches predominate, reflecting the interest in their use where they enable sample collection which is not feasible with standard approaches or to derive richer data sets.

Liquid chromatography-high resolution mass spectrometry for broad-spectrum drug screening of dried blood spot as microsampling procedure.

BACKGROUND: Hyphenation of liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) offers the potential to develop broad-spectrum screening procedures from low volumes of biological matrices. In parallel, dried blood spot (DBS) has become a valuable tool in the bioanalysis landscape to overcome conventional blood collection issues. Herein, we demonstrated the applicability of DBS as micro-sampling procedure for broad-spectrum toxicological screening. METHODS: A method was developed on a HRMS system in data dependant acquisition (DDA) mode using an extensive inclusion list to promote collection of relevant data. 104 real toxicology cases were analysed, and the results were cross-validated with one published and one commercial screening procedures. Quantitative MRM analyses were also performed on identified substances on a triple quadrupole instrument as a complementary confirmation procedure. RESULTS: The method showed limits of identification (LOIs) in appropriateness with therapeutic ranges for all the classes of interest. Applying the three screening approaches on 104 real cases, 271 identifications were performed including 14 and 6 classes of prescribed and illicit drugs, respectively. Among the detected substances, 23% were only detected by the proposed method. Based on confirmatory analyses, we demonstrated that the use of blood micro-samples did not impair the sensitivity allowing more identifications in the low concentration ranges. CONCLUSION: A LC-HRMS assay was successfully developed for toxicological screening of blood microsamples demonstrating a high identification power at low concentration ranges. The validation procedure and the analysis of real cases demonstrated the potential of this assay by supplementing screening approaches of reference.

Association of reported fish intake and supplementation status with the omega-3 index.

BACKGROUND: An Omega-3 Index (O3I; EPA+DHA as a % of erythrocyte total fatty acids) in the desirable range (8%-12%) has been associated with improved heart and brain health. OBJECTIVE: To determine the combination of fish intake and supplement use that is associated with an O3I of >8%. DESIGN: Two cross-sectional studies comparing the O3I to EPA+DHA/fish intake. PARTICIPANTS/SETTING: The first study included 28 individuals and assessed their fish and EPA+DHA intake using both a validated triple-pass 24-hr recall dietary survey and a single fish-intake question. The second study used de-identified data from 3,458 adults (84% from US) who self-tested their O3I and answered questions about their fish intake and supplement use. STATISTICAL ANALYSES PERFORMED: Study 1, chi-squared, one-way ANOVA, and Pearson correlations were computed. In Study 2, multi-variable regression models were used to predict O3I levels from reported fish/supplement intakes. RESULTS: The mean ±â€¯SD O3I was 4.87 ±â€¯1.32%, and 5.99 ±â€¯2.29% in the first and second studies, respectively. Both studies showed that for every increase in fish intake category the O3I increased by 0.50-0.65% (p < 0.0001). In the second study, about half of the population was taking omega-3 supplements, 32% reported no fish intake and 17% reported eating fish >2 times per week. Taking an EPA+DHA supplement increased the O3I by 2.2% (p < 0.0001). The odds of having an O3I of ≥8% were 44% in the highest intake group (≥3 servings/week and supplementation) and 2% in the lowest intake group (no fish intake or supplementation); and in those consuming 2 fish meals per week but not taking supplements (as per recommendations), 10%. CONCLUSIONS: Current AHA recommendations are unlikely to produce a desirable O3I. Consuming at least 3 fish servings per week plus taking an EPA+DHA supplement markedly increases the likelihood of achieving this target level.

Testing for hepatitis C virus infection in UK prisons: What actually happens?

Prisons are a key demographic in the drive to eradicate hepatitis C virus (HCV) as a major public health threat. We have assessed the impact of the recently introduced national opt-out policy on the current status of HCV testing in 14 prisons in the East Midlands (UK). We analysed testing rates pre- and post-introduction of opt-out testing, together with face-to-face interviews with prison healthcare and management staff in each prison. In the year pre-opt-out, 1972 people in prison (PIP) were tested, compared to 3440 in the year following opt-out. From July 2016 to June 2017, 2706 people were tested, representing 13.5% of all prison entrants (median 16.6%, range 7.6%-40.7%). Factors correlating with testing rates were as follows: pre-admission location of the PIP (another prison or the community, OR 2.2, 95% CI 1.9-2.3, P < 0.001); whether the PIP could access health care independently of prison officers (OR 1.7, 95% CI 1.5-1.8, P < 0.001); the absence of out-reach services for HCV treatment (OR 1.3, 95% CI 1.2-1.5, P < 0.001), whether >50% of PIP reported ease of access to a nurse (OR 2.0, 95% CI 1.8-2.2, P < 0.001), and whether prison health care was supplied by private or NHS providers (OR 1.3, 95% CI 1.2-1.5, P < 0.001). Testing rates remained far below the minimum national opt-out target of 50%. Inadequacy of healthcare facilities and constraints imposed by adherence to prison regimens were cited by healthcare and management staff at all prisons. Without radical change, the prison estate may be intrinsically incapable of supporting NHSE to deliver the HCV elimination strategy.

Phenylalanine and tyrosine measurements across gestation by tandem mass spectrometer on dried blood spot cards from normal pregnant women.

PURPOSE: Maternal phenylketonuria (MPKU) requires strict control of phenylalanine (Phe) and supplemental tyrosine (Tyr). Monitoring during pregnancy using dried blood spot (DBS) cards by tandem mass spectrometry (MS/MS) is now standard practice, however there are no Phe and Tyr reference ranges for DBS MS/MS method in healthy pregnant women. METHODS: DBS cards (63-1364 days in storage) from healthy women with singleton pregnancies were analyzed by MS/MS. Three hundred ninety DBS cards from 170 pregnancies (5/1-39/6 weeks' gestation), were tested. RESULTS: Both Phe and Tyr levels declined from the first trimester (Phe: 36.2 +/- 10.6; Tyr 25.7 +/- 9.7 µmol/L) to the second trimester (Phe 33.4+/-9.3; Tyr 21.7+/- 6.7 µmol/L) and remained stable in the third trimester (Phe 32.3 +/- 8.7; Tyr 21.0 +/- 6.6 µmol/L). Phe and Tyr levels declined over time since collection (Phe: 0.004 µmol/L per day; Tyr 0.002 µmol/L). Nomograms by gestational age were created using raw data and data adjusted for time from sample collection. Reference ranges by trimester are provided. CONCLUSIONS: Both Phe and Tyr decline quickly during the first trimester and remain relatively constant over the second and third trimesters. These nomograms will provide a valuable resource for care of MPKU.