A strategy of accuracy quantification by extending the concentration monitoring coverage based on online double collision energy of ultra-high performance liquid chromatography tandem mass spectrometry: The pharmacokinetics of Toddalia asiatica as a case study.

To facilitate the safety, efficacy and rationality of clinical application of traditional Chinese medicines (TCMs), pharmacokinetic research played an indispensable role. The key challenge during pharmacokinetic investigation lied at the substantial fluctuation of compound concentrations in the plasma over the course of absorption. Taking the pharmacokinetics of six compounds after administration of Toddalia asiatica (TA) as an example, an efficient strategy was established by introducing the online double collision energy (ODCE) into the quantification process applying ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). During the analytical program, double collision energy (DCE) was optimized to establish the dual calibration curve (DCC) with large concentration monitoring coverage (CMC) for meeting the wide content range of certain target compounds. Method validation test was performed in terms of linearity, precision, sensitivity, matrix effect, recovery, etc. The results displayed that the CMC of todarolactone with high exposure in plasma was extended from 1.25-2,500 ng/mL to 1.25-125,000 ng/mL. Furthermore, a rapid UHPLC-MS/MS method integrated with ODCE was successfully applied to the determination of six compounds in rat plasma, revealing an extremely high plasma concentration of todarolactone (16,662 ng/mL). This strategy could expand the range of quantification while retaining extraordinary sensitivity. Consequently, it could be a fit-for-purpose strategy to quantify compounds over a wide concentration range for in vivo process monitoring.

Safety, pharmacokinetics and CNS distribution of tralesinidase alfa administered via intracerebroventricular infusion to juvenile cynomolgus monkeys.

Mucopolysaccharidosis Type IIIB (MPS IIIB) is an ultrarare, fatal pediatric disease with no approved therapy. It is caused by mutations in the gene encoding for lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Tralesinidase alfa (TA) is a fusion protein comprised of recombinant NAGLU and a modified human insulin-like growth factor 2 that is being developed as an enzyme replacement therapy for MPS IIIB. Since MPS IIIB is a pediatric disease the safety/toxicity, pharmacokinetics and biodistribution of TA were evaluated in juvenile non-human primates that were administered up to 5 weekly intracerebroventricular (ICV) or single intravenous (IV) infusions of TA. TA administered by ICV slow-, ICV isovolumetric bolus- or IV-infusion was well-tolerated, and no effects were observed on clinical observations, electrocardiographic or ophthalmologic parameters, or respiratory rates. The drug-related changes observed were limited to increased cell infiltrates in the CSF and along the ICV catheter track after ICV administration. These findings were not associated with functional changes and are associated with the use of ICV catheters. The CSF PK profiles were consistent across all conditions tested and TA distributed widely in the CNS after ICV administration. Anti-drug antibodies were observed but did not appear to significantly affect the exposure to TA. Correlations between TA concentrations in plasma and brain regions in direct contact with the cisterna magna suggest glymphatic drainage may be responsible for clearance of TA from the CNS. The data support the administration of TA by isovolumetric bolus ICV infusion to pediatric patients with MPS IIIB.

Method validation of multi-element panel in whole blood by inductively coupled plasma mass spectrometry (ICP-MS).

Background: Analytical methods to measure trace and toxic elements are essential to evaluate exposure and nutritional status. A ten-element panel was developed and validated for clinical testing in whole blood. Retrospective data analysis was conducted on patient samples performed at ARUP Laboratories. Methods: A method was developed and validated to quantify ten elements in whole blood by ICP-MS. Fifty microliters of sample were extracted with 950 µL of diluent containing 1 % ammonium hydroxide, 0.1 % Triton X-100, 1.75 % EDTA along with spiked internal standards. Four calibrators were used for each element and prepared in goat blood to match the patient specimen matrix. Samples were analyzed with an Agilent 7700 ICP-MS with a Cetac MVX 7100 µL Workstation autosampler. Results: The assay was linear for all elements with inter- and intra-assay imprecision less than or equal to 11% CV at the low end of the analytical measurement range (AMR) and less than or equal to 4% CV at the upper end of the AMR for all elements. Accuracy was checked with a minimum of 40 repeat patient samples, proficiency testing samples, and matrix-matched spikes. The linear slopes for the ten elements ranged from 0.94 to 1.03 with intercepts below the AMR and R2 ranging from 0.97 to 1.00. Conclusions: The multi-element panel was developed to analyze ten elements in whole blood to unify the sample preparation and increase batch run efficiency. The improved analytical method utilized matrix-matched calibrators for accurate quantification to meet regulatory requirements. The assay was validated according to guidelines for CLIA-certified clinical laboratories and was suitable for clinical testing to assess nutritional status and toxic exposure.

Development of an automated chemiluminescent immunoassay for cancer antigen 72-4 and the evaluation of its analytical performance.

Objectives: Cancer antigen (CA) 72-4 assay is widely used for monitoring gastric and ovarian cancers. The antigen is a mucin-like, tumor-associated glycoprotein known as TAG-72. It has been identified and characterized using two different monoclonal antibodies, CC49 and B72.3, which recognize its glycochain epitopes, Galß(1-3) sialyl-Tn and sialyl-Tn antigens, respectively. This study describes the quantitative analytical performance of a newly developed CA 72-4 assay, ARCHITECT CA 72-4. Design: and Methods: The ARCHITECT CA 72-4 assay was developed using the ARCHITECT i2000SRs and three ARCHITECT i1000SRs. The assay performance was evaluated based on guidance from CLSI (Clinical and Laboratory Standards Institute) and correlation against Elecsys CA 72-4. Results: In the total precision study, the minimum coefficient of variation (CV) for Control/Panel samples over 4 U/mL was 1.1%. The measuring interval was from 0.95 to 200 U/mL with good linearity; and limits of blank (LoB), detection (LoD), and quantitation (LoQ) were 0.09, 0.18, and 0.95 U/mL, respectively. High dose hook effect; differences among specimen tube types; and interference of common drugs, potential cross-reactants, and endogenous substances were not observed. Significantly, this assay has high biotin tolerance at 4875 mg/mL and correlates well with the Elecys CA 72-4 assay (correlation coefficient: 0.95). Conclusions: ARCHITECT CA 72-4 is a highly sensitive and precise assay for CA 72-4 measurement in human sera and plasma.

Identification of a frit-related sample carryover in newborn screening by tandem mass spectrometry.

The need for high-throughput analysis of multiple analytes for inborn errors of metabolism in newborn screening (NBS) has led to the introduction of tandem mass spectrometry (MS/MS) into the NBS laboratory. In a flow-injection analysis (FIA), the predominant MS/MS method utilized for NBS, samples are introduced directly into the mass spectrometer without chromatographic separation. When a high-throughput FIA-based MS/MS method is implemented on newer generations of mass spectrometers with increased sensitivity, the risk of carryover and contamination increases. In the present study, we report the carryover of ornithine identified during the implementation of the NeoBase™ 2 (PerkinElmer) non-derivatized kits on the Xevo-TQD platform (Waters Corporation) and describe the source of the carryover, which was traced to the stainless-steel frit-type inline filter. Furthermore, a possible compound-dependent interaction with the stainless-steel frit is suggested based on the structure of ornithine and its effect on separation techniques. Investigation and mitigation of carryover can be a time and resource consuming process, and to this end, our report on identification of a stainless-steel frit as the source of delayed elution and carryover of ornithine should be recognized as a rare, albeit possible source of carryover in FIA-MS/MS methods adopted for NST.

Validation of a quantitative multiplex LC-MS/MS assay of carvedilol, enalaprilat, and perindoprilat in dried blood spots from heart failure patients and its cross validation with a plasma assay.

Introduction: Adherence to medication is an important determinant of outcomes in chronic diseases like heart failure. Drug assays provide objective adherence biomarkers. Dried blood spots (DBS) are appealing samples for drug assays due to less demanding transportation and storage requirements. Objectives: To analytically validate a LC-MS/MS method for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS and evaluate the feasibility of using the method as an adherence determining assay. To validate the assay further clinically by establishing correlation and agreement between plasma and DBS samples from a pharmacokinetic pilot study. Methods: The method was validated over a concentration range of 1.00-200 ng/mL according to FDA guidelines. Adherence tracking ability of the assay was evaluated using a pharmacokinetic pilot study. Correlation and agreement were evaluated through Deming regression and Bland-Altman analysis, respectively. Results: Accuracy, precision, selectivity, and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested. Stability of the analytes in the matrix and throughout sample processing was proven. The full range of concentrations of the pharmacokinetic pilot study could be quantified for enalaprilat, but not for carvedilol and perindoprilat. The difference between the observed and calculated plasma concentrations was less than 20 % of their mean for >67 % of samples for all analytes. Conclusions: The assay is suitable as a screening tool for carvedilol and perindoprilat, while suitable as an adherence determining assay for enalaprilat. Equivalence between observed and predicted plasma concentrations proves DBS and plasma concentrations can be used interchangeably.

LC-MS/MS method for simultaneous quantification of ten antibiotics in human plasma for routine therapeutic drug monitoring.

Background: Optimizing antimicrobial therapy to attain drug exposure that limits the emergence of resistance, effectively treats the infection, and reduces the risk of side effects is of a particular importance in critically ill patients, in whom normal functions are augmented or/and are infected with pathogens less sensitive to treatment. Achievement of these goals can be enhanced by therapeutic drug monitoring (TDM) for many antibiotics. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is presented here for simultaneous quantification of ten antimicrobials: cefazolin (CZO), cefepime (CEP), cefotaxime (CTA), ceftazidime (CTZ), ciprofloxacin (CIP), flucloxacillin (FLU), linezolid (LIN), meropenem (MER), piperacillin (PIP) and tazobactam (TAZ) in human plasma. Methods: Plasma samples were precipitated with acetonitrile and injected into the LC-MS/MS. Chromatographic separation was on a Waters Acquity BEH C18 column. Compounds were eluted with water and acetonitrile containing 0.1 % formic acid, using a gradient (0.5-65 % B), in 3.8 min. The flow rate was 0.4 mL/min, and the run time was 5.8 min. Results: The calibration curves were linear across the tested concentration ranges (0.5-250, CZO, CEP, CTA, CTZ and FLU; 0.2-100, MER and TAZ; 0.1-50, CIP and LIN and 1-500 mg/L, PIP). The intra and inter-day imprecision was < 11 %. Accuracy ranged from 95 to 114 %. CTZ and MER showed ionization suppression while CIP showed ionization enhancement, which was normalized with the use of the internal standard. Conclusion: An LC-MS/MS method for simultaneous quantification of ten antimicrobials in human plasma was developed for routine TDM.

Postmortem sampling time effect on toxicity biomarkers in rats exposed to an acute lethal methomyl dose.

Regulations often are imposing long postmortem times before autopsy leading to certain toxicity-unrelated changes in biomarkers, which in turn may affect the reliability of toxicity evaluation during forensic investigations. Since methomyl pesticide shows significant toxicity and is frequently encountered in poisoning cases, the current study evaluated different parameters in methomyl intoxicated rats at three different postmortem intervals (Hour 0, Hour 3 and Hour 6). Eighteen adult Sprague Dawley rats were poisoned with methomyl to simulate actual methomyl poisoning cases. The time of death was assigned as Hour 0. The animals were divided into 3 groups (n = 6) to collect blood and tissue samples at the selected time points. Body weight, relative organ weight, protein concentration, methomyl concentration and acetylcholinesterase activity (AChE) were assessed in blood and different tissues (liver, spleen, kidney, brain, eye, and bone marrow) to evaluate the effect of postmortem sampling time. Outcomes revealed significant decreases in methomyl concentration in blood and bone marrow with advanced sampling time (P < 0.001). Similarly, there were significant reductions in AChE activity in the kidney (P < 0.01), while the enzyme activity significantly increased in brain samples (P < 0.05). Findings illustrated the importance of sampling time in toxicity studies because it could alter experimental results and impact consequent interpretations, as well as it may alter postmortem biomarkers in related forensic cases.

Development and validation of a paper spray mass spectrometry method for the rapid quantitation of remdesivir and its active metabolite, GS-441524, in human plasma.

Introduction: Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2). It is currently the only FDA-approved antiviral agent for the treatment of individuals with COVID-19 caused by SARS-CoV-2. However, remdesivir pharmacokinetics/pharmacodynamics (PK/PD) and toxicity data in humans are extremely limited. It is imperative that precise analytical methods for the quantification of remdesivir and its active metabolite, GS-441524, are developed for use in further studies. We report, herein, the first validated anti-viral paper spray-mass spectrometry (PS-MS/MS) assay for the quantification of remdesivir and GS-441524 in human plasma. We seek to highlight the utility of PS-MS/MS technology and automation advancements for its potential future use in clinical research and the clinical laboratory setting. Methods: Calibration curves for remdesivir and GS-441524 were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Briefly, plasma samples were spotted on a paper substrate contained within pre-manufactured plastic cassette plates, and the spots were dried for 1 h. The samples were then analyzed directly for 1.2 min utilizing PS-MS/MS. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer utilizing automated technology. Results: The calibration ranges were 20 - 5000 and 100 - 25000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process. Conclusions: PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir and GS-441524, which may be useful for clinical PK/PD, therapeutic drug monitoring (TDM), and toxicity assessment, particularly during the current COVID-19 pandemic and future viral outbreaks.

Multiplexed quantification of insulin and C-peptide by LC-MS/MS without the use of antibodies.

Introduction: The measurement of insulin and C-peptide provides a valuable tool for the clinical evaluation of hypoglycemia. In research, these biomarkers are used together to better understand hyperinsulinemia, hepatic insulin clearance, and beta cell function. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an attractive approach for the analysis of insulin and C-peptide because the platform is specific, can avoid certain limitations of immunoassays, and can be multiplexed. Previously described LC-MS/MS methods for the simultaneous quantification of insulin and C-peptide measure the intact analytes and most have relied on immunoaffinity enrichment. These approaches can be limited in terms of sensitivity and interference from auto-antibodies, respectively. We have developed a novel method that does not require antibodies and uses proteolytic digestion to yield readily ionizable proteotypic peptides that enables the sensitive, specific, and simultaneous quantitation of insulin and C-peptide. Methods: Serum samples were precipitated with acetonitrile. Analytes were enriched using solid phase extraction and then digested with endoproteinase Glu-C. Surrogate peptides for insulin and C-peptide were analyzed using targeted LC-MS/MS. Results: Inter-day imprecision was below 20 %CV and linearity was observed down to the lower limit of quantitation for both analytes (insulin = 0.09 ng/mL, C-peptide = 0.06 ng/mL). Comparison to a commercially available insulin immunoassay (Beckman Coulter UniCel DxI 600 Access) revealed a 30% bias between methods. Conclusion: A novel LC-MS/MS method for the simultaneous analysis of insulin and C-peptide using Glu-C digestion was developed and evaluated. A detailed standard operating procedure is provided to help facilitate implementation in other laboratories.

Biotechnological formation of dairy flavor inducing δ-lactones from vegetable oil.

Agroscope Culture Collection was screened to identify bacterial strains effective in production of dairy flavor inducing lactones using grapeseed oil as a substrate. Lentilactobacillus parafarraginis FAM-1079, Lactococcus lactis subsp. lactis FAM-17918, and L. lactis subsp. lactis biovar diacetylactis FAM-22003 showed the most efficient formation of targeted δ-lactones. The application of sublethal heat stress significantly increased target lactone production. The most profound improvement was for L. lactis subsp. lactis biovar diacetylactis where δ-octadecalactone generation was improved by factor of 9. The pre-fermentation step as well as growth phase in which bacteria are harvested did not have a significant impact on lactones yield. The lactone production process from vegetable oil developed in this study offers a new way of developing a natural flavor ingredient for incorporation into plant-based products.

A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease.

Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used. Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma. Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays. Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean. Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

Quantitation of non-derivatized free amino acids for detecting inborn errors of metabolism by incorporating mixed-mode chromatography with tandem mass spectrometry.

Introduction: Amino acids are critical biomarkers for many inborn errors of metabolism, but amino acid analysis is challenging due to the range of chemical properties inherent in these small molecules. Techniques are available for amino acid analysis, but they can suffer from long run times, laborious derivatization, and/or poor resolution of isobaric compounds. Objective: To develop and validate a method for the quantitation of a non-derivatized free amino acid profile in both plasma and urine samples using mixed-mode chromatography and tandem mass spectrometry. Methods: Chromatographic conditions were optimized to separate leucine, isoleucine, and allo-isoleucine and maintain analytical runtime at less than 15 min. Sample preparation included a quick protein precipitation followed by LC-MS/MS analysis. Matrix effects, interferences, linearity, carryover, acceptable dilution limits, precision, accuracy, and stability were evaluated in both plasma and urine specimen types. Results: A total of 38 amino acids and related compounds were successfully quantitated with this method. In addition, argininosuccinic acid was qualitatively analyzed. A full clinical validation was performed that included method comparison to a reference laboratory for plasma and urine with Deming regression slopes ranging from 0.38 to 1.26. Conclusion: This method represents an alternative to derivatization-based methods, especially in urine samples where interference from metabolites and medications is prevalent.

Assessing the microcystins concentration through optimized protein phosphatase inhibition assay in environmental samples.

Protein phosphatase (PPase) inhibition assay (PPIA) is widely used to analyze the concentration of microcystins (MCs) because it is comparatively less expensive and faster than other assays. This study aimed to optimize the PPIA by determining a suitable reaction terminator and an optimal methanol concentration in the sample. The most suitable reaction time was 90 min, with the corresponding methanol concentration in the sample being 15% or less. When p-nitrophenyl phosphate (pNPP) was used as a substrate, copper chloride solution was suitably used as a reaction terminator, and when 4-methylumbelliferyl phosphate (MUP) was used, a glycine buffer not only increased the measurement sensitivity of the reaction product but also terminated the enzymatic reaction. When PPase 1 and MUP were used as an enzyme and a substrate, respectively, the limit of quantitation for MC-leucine/arginine (LR) was 0.02 µg/L, whereas it was 0.1 µg/L when pNPP was used as a substrate. The proposed method facilitated the measurement of MC-LR concentration without additional pretreatments, such as concentration or purification; therefore, this method was suitable and feasible for the continuous monitoring of MCs in drinking water.

Pharmacokinetics and analytical determination of acyclovir in Asian elephant calves (Elephas maximus).

A therapeutic regimen that includes antiviral drugs is critical for the survival of Asian elephant (Elephas maximus) calves infected with elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD), with acyclovir showing considerable promise. The purpose of this study was to determine the pharmacokinetics and bioavailability of acyclovir following intravenous (IV) and oral (PO) administration in Asian elephants. A single dose of acyclovir (15 mg/kg, IV or 45 mg/kg, PO) was administered to four healthy elephant calves, with a minimum 2-week washout period between treatments. Serial plasma samples were collected after each injection for acyclovir analysis using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Maximum plasma acyclovir concentrations were 27.02 ± 6.79 µg/mL at 0.94 ± 0.31 h after IV administration, and 1.45 ± 0.20 µg/mL at 3.00 ± 0.70 h after PO administration. The half-life of the elimination phase (T1/2) was 5.84 ± 0.74 and 8.74 ± 2.47 h after IV and PO administration, respectively. After IV administration, acyclovir concentrations were higher than the half-maximal inhibitory concentration (IC50) of those found for herpes simplex virus (HSV) 1 and 2 in humans, and equid alpha herpesvirus-1 (EHV-1) for at least 12 h. By contrast, the bioavailability of oral administration was low, only 6.03 ± 0.87%, so higher doses by that route likely are needed to be effective. Due to the high concentration of plasma acyclovir after IV administration, the dose may need to be adjusted to prevent any negative side effects.

Progress on the investigation and monitoring of marine phycotoxins in China.

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin ß-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

A novel method development and validation for determination of 2,4,6-Trinitrotoluene and its metabolites on LC-MS/MS.

LC-MS/MS has recently emerged as the best practice for simultaneous analysis of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites. We have developed and validated an LC-MS/MS method for simultaneous quantification of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites 4-ADNT, 2-ADNT, 2,4-DNT, and 2,6-DNT in urine samples. These four metabolites were acid hydrolyzed using 1 mL of urine followed by extraction using n-Hexane and ethyl acetate as an extracting solvent. Separation was achieved by centrifugation, and the supernatant was dried under nitrogen, reconstituted with water and acetonitrile, and then filtered. Chromatographic separation was achieved on Agilent Poroshel 120 EC-C18 column (2.1 mm × 75 mm × 2.7 µm) utilizing two mobile phases 0.1% formic acid in water and 0.1% formic acid in acetonitrile in gradient flow. The validated AMR of TNT and its metabolites was 7.8-1000 ng/mL. The method showed an excellent correlation (>0.99) for TNT and its metabolites. Accuracy and within/between day precision of TNT and its metabolites were within ±15%. The integrity of diluted samples was maintained for each dilution factor. The method was found stable after storage and freeze-thaw cycle. The presented method can be used for TNT screening in occupationally exposed ordnance factory workers.

Assay of ethanol and congener alcohols in serum and beverages by headspace gas chromatography/mass spectrometry.

The analysis of ethanol and of its congeners in blood plays an important role in forensic cases, especially when allegations are made that alcohol has been consumed after an accident. In alcoholic beverages, congener alcohols are by-products and are generated during fermentation. The assay of these compounds in serum samples and beverages has been previously performed using headspace-gas chromatography-flame ionization detection methods (HS-GC-FID). As an alternative, a robust headspace-gas chromatography-mass spectrometry (HS-GC-MS) procedure was developed and validated, which has the following advantages:•Simultaneous determination of ethanol, congener alcohols and other endogenous substances.•Reduction of matrix interference by increasing selectivity and specificity.•Clear separation of the positional isomers 3-methyl-1-butanol and 2-methyl-1-butanol.

Validation of a simple liquid chromatography coupled to tandem mass spectrometry method for the simultaneous determination of tacrolimus, sirolimus, everolimus and cyclosporin A in dried matrix on paper discs.

INTRODUCTION: Due to its high specificity and sensitivity, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard method for immunosuppressant quantification in therapeutic drug monitoring. In this context, dried blood spots (DBS) have become a promising strategy as a sample collection procedure. Although the advantages of DBS over venipuncture are well known, this approach has limitations that strongly influence the acceptance of analytical results. Among them, the most important is hematocrit (Ht). The easiest way of overcoming this problem is by analyzing complete spots. In this strategy, called dried matrix on paper discs (DMPD), blood is volumetrically applied on pre-punched discs. OBJECTIVES: To validate an LC-MS/MS method for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A using DMPD. METHODS: The procedure was validated according to international guidelines using a commercial kit. The following performance parameters were evaluated: selectivity, carryover, linearity, accuracy, precision, lower limit of quantitation, relative recovery, commutability and stability. In addition, a method comparison study was performed to evaluate the clinical influence of Ht on the results. RESULTS: All performance parameters were within acceptance criteria and, hence, it was determined that the validated method is fit for the intended purpose. Likewise, calculated bias values on medical decision levels showed that there was no clinical influence of Ht on the results. CONCLUSION: Unlike other similar methodologies that have been published, here, a simple method has been fully validated. This is the first LC-MS/MS methodology adapting a commercial kit to use DMPD as a sampling strategy.

Analysis of 2-methylcitric acid, methylmalonic acid, and total homocysteine in dried blood spots by LC-MS/MS for application in the newborn screening laboratory: A dual derivatization approach.

Inborn errors of propionate, cobalamin and methionine metabolism are targets for Newborn Screening (NBS) in most programs world-wide, and are primarily screened by analyzing for propionyl carnitine (C3) and methionine in dried blood spot (DBS) cards using tandem mass spectrometry (MS/MS). Single-tier NBS approaches using C3 and methionine alone lack specificity, which can lead to an increased false-positive rate if conservative cut-offs are applied to minimize the risk of missing cases. Implementation of liquid chromatography tandem mass spectrometry (LC-MS/MS) second-tier testing for 2-methylcitric acid (MCA), methylmalonic acid (MMA), and homocysteine (HCY) from the same DBS card can improve disease screening performance by reducing the false-positive rate and eliminating the need for repeat specimen collection. However, DBS analysis of MCA, MMA, and HCY by LC-MS/MS is challenging due to limited specimen size and analyte characteristics leading to a combination of low MS/MS sensitivity and poor reverse-phase chromatographic retention. Sufficient MS response and analytical performance can be achieved for MCA by amidation using DAABD-AE and by butylation for MMA and HCY. Herein we describe the validation of a second-tier dual derivatization LC-MS/MS approach to detect elevated MCA, MMA, and HCY in DBS cards for NBS. Clinical utility was demonstrated by retrospective analysis of specimens, an interlaboratory method comparison, and assessment of external proficiency samples. Imprecision was <10.8% CV, with analyte recoveries between 90.2 and 109.4%. Workflows and analytical performance characteristics of this second-tier LC-MS/MS approach are amenable to implementation in the NBS laboratory.