Contribution of methamphetamine and insulin to the death of a woman suffering from type I diabetes - which played the greater role?

This report presents a fatal case of a young female Type I diabetic patient who developed convulsions and loss of consciousness after taking methamphetamine and spending some time in a dance club. During the convulsions, she was given sugar and when no response occurred, her boyfriend who was not experienced in the use of insulin administered a dose of insulin to her. The woman lost consciousness and died despite the efforts of the emergency service. A biochemical analysis revealed a high level of insulin (196.67 mU/L) and low levels of glucose (2.96 mmol/L) and C-peptide (26 pmol/L). Toxicological analysis revealed a methamphetamine concentration of 389 ng/mL and an amphetamine concentration of 19 ng/mL. The forensic perspective of the difficult determination of the contribution of each of the factors to the death, i.e., the pre-existing medical condition (Type I diabetes), the use of methamphetamine, the physical exertion at the dance club, and, finally, the non-indicated administration of insulin, is discussed. The ruling of the court is also reported.

Liquid chromatography-tandem mass spectrometry for determination of fingolimod and its active metabolite fingolimod phosphate in whole blood of patients with multiple sclerosis.

Fingolimod is an oral drug for the escalation of treatment of relapsing-remitting multiple sclerosis in patients with persistent disease activity on first-line drugs or in patients with rapidly progressive severe relapsing-remitting multiple sclerosis. An ultra-high-performance liquid chromatography-tandem mass spectrometry method for determining the concentrations of fingolimod and its active metabolite fingolimod phosphate in whole blood has been developed and validated. The advantages of this method are the easy, fast and cheap sample preparation using protein precipitation from blood with a mixture of acetonitrile-methanol (40:60, v/v). Chromatographic separation was performed on a ultra-high performance liquid chromatography BEH C18 1.7 µm (100 × 2.1 mm) column. Two modes of ionization, electrospray ionization and atmospheric pressure chemical ionization, were tested and compared. For validation, the electrospray ionization mode was chosen. As internal standard, isotopically labeled fingolimod-D4 was used to quantify the analytes. The method was validated according to the rules of the European Medicines Agency. The coefficients of variation for fingolimod were in the range of 1.13-11.88%, and the recovery was 98.80-106.00%. The coefficients of variation for fingolimod phosphate were in the range of 2.73-9.31%, and the recovery was 90.08-107.00%. The method is quite easy and fast and can be used for routine analysis.

Fast and Easy Simultaneous Determination of Riboflavin, Folic Acid, All-Trans-Retinol and α-Tocopherol in Human Serum by LC/MS/MS for Bariatric Patients.

The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry (MS) detection. Vitamin analysis was performed using an ultra performance liquid chromatography combined with tandem MS. The compounds were separated on a BEH C18 RP column (2.1 × 100 mm, 1.7 µm) using a gradient elution with an analysis time of 10 min. Sample preparation included protein precipitation with ethanol. The concentration range in human serum was as follows: riboflavin 5-1000 nmol/L, folic acid 2.5-250 nmol/L, α-tocopherol 0.5-100 µmol/L and all-trans-retinol 25-2500 nmol/L. Accuracy and precision were validated according to Food and Drug Administration guidelines, with coefficients of variation ranging from 3.1-11.7% and recoveries from 94.4-107.5%. Routine monitoring of the complex range of vitamins in bariatric medicine is still not common. This is despite the fact that patients are at risk for glitch deficits, especially of a neurological nature. An analytical method that allows for the complex measurement of both water-soluble and fat-soluble vitamins is important and necessary for the clinical monitoring of bariatric patients. The method we have described could benefit both clinical practice and nutritional research.

Liquid chromatography-tandem mass spectrometry methods for quantification of total and free antibiotic concentrations in serum and exudate from patients with post-sternotomy deep sternal wound infection receiving negative pressure wound therapy.

BACKGROUND: Systemically administered antibiotics are thought to penetrate the wounds more effectively during negative pressure wound therapy (NPWT).To test this hypothesis total and free antibiotic concentrations were quantified in serum and wound exudate. METHODS: UHPLC-MS/MS methods were developed and validated for the determination of ceftazidime, cefepime, cefotaxime, cefuroxime, cefazolin, meropenem, oxacillin, piperacillin with tazobactam, clindamycin, ciprofloxacin, sulfamethoxazole/trimethoprim (cotrimoxazole), gentamicin, vancomycin, and linezolid. The unbound antibiotic fraction was obtained by ultrafiltration using a Millipore Microcon-30kda Centrifugal Filter Unit. Analysis was performed on a 1.7-µm Acquity UPLC BEH C18 2.1 × 100-mm column with a gradient elution. RESULTS: The validation was performed for serum, exudates and free fractions. For all matrices, requirements were met regarding linearity, precision, accuracy, limit of quantitation, and matrix effect. The coefficient of variation was in the range of 1.2-13.6%.and the recovery 87.6-115.6%, respectively. Among the 29 applications of antibiotics thus far, including vancomycin, clindamycin, ciprofloxacin, oxacillin, cefepime, cefotaxime, cotrimoxazole, and gentamicin, total and free antibiotic concentrations in serum and exudate were correlated. CONCLUSION: This method can accurately quantify the total and free concentrations of 16 antibiotics. Comparison of concentration ratios between serum and exudates allows for monitoring individual antibiotics' penetration capacity in patients receiving NPWT.

Liquid chromatography - tandem mass spectrometry method for determination of natalizumab in serum and cerebrospinal fluid of patients with multiple sclerosis.

Natalizumab is a humanized recombinant monoclonal IgG4 antibody used in the treatment of multiple sclerosis. Commonly used methods for natalizumab and anti-natalizumab antibodies quantification are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Measurement of therapeutic monoclonal antibodies can be challenging due to the resemblance to human plasma immunoglobulins. Recent developments in mass spectrometry enables to analyze vast variety of large protein molecules. The aim of this study was to develop a LC-MS/MS method for determining natalizumab in human serum and cerebrospinal fluid (CSF) and apply it to clinical settings. For successful quantification, it was necessary to find specific sequences of peptides in natalizumab. This immunoglobulin was treated with dithiothreitol and iodoacetamide, cleaved with trypsin into short specific peptides and determined on a UPLC-MS/MS system. An Acquity UPLC BEH C18 column at 55 °C and gradient elution was used for analysis. Intra- and interassay accuracies and precisions were tested at four concentration levels. Precision was determined by coefficients of variation and was in the range of 0.8-10.2 %, with accuracy in the range of 89.8-106.4 %. The concentration of natalizumab in patient samples ranged from 1.8 to 193.3 µg/mL. The method was validated according to the European Medicines Agency (EMA) guideline, met all acceptance criteria for accuracy and precision, and is suitable for clinical applications. In comparison to immunoassay, which can be elevated by cross-reaction with endogenous immunoglobulins, the results of developed LC-MS/MS method are more accurate and specific.

Liquid chromatography-tandem mass spectrometry method for determination of total and free teriflunomide concentration in serum of patients with multiple sclerosis.

Teriflunomide belongs to disease-modifying drugs and is used in treatment of multiple sclerosis. According to in vitro studies more than 99.4 % of drug is binding to plasma proteins and only less than 1 % is free for clinical activity. The rapid and simple ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was developed and validated for determination of total and free teriflunomide (TFM) in serum of patients with multiple sclerosis. To determine the total teriflunomide samples were precipitated with a precipitation reagent consisting of 11 % solution of ZnSO4 in acetonitrile/methanol (40:60, v/v). To determine the free fraction of teriflunomide, an ultracentrifugation method was used. The analysis was performed on a UPLC system connected to a XEVO TQ-XS mass spectrometer. Chromatographic separation was carried out on an Acquity UPLC BEH C18 1.7 µm (100 × 2.1 mm) column heated to 30 °C and teriflunomide-D4 was used as an internal standard. Ionization was performed by electrospray in negative ion mode. The developed methods were validated according to the rules of the European Medicines Agency (EMA) for the analytical method validation of bioanalytical methods. The coefficients of variation were in the range of 0.53-14.84 % and the recovery 97.92-108.33 %, respectively. Share of free teriflunomide was 0.15-0.40 % (mean 0.25 ± 0.05 %) of total teriflunomide and there was a significant correlation between free and total teriflunomide r2 = 0.9083 (p < 0.0001). This newly developed method allows the rapid and easy determination of the teriflunomide concentration with high sensitivity and can be applied to clinical samples of patients with multiple sclerosis.

New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations.

A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50µL serum was precipitated using 20µL 33% trichloroacetic acid and 0.5mol/L NH4OH was added to increase pH before analysis. A RP BEH C18, 1.7µm, 2.1×50mm column maintained at 30°C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5±19.1, 150.2±48.4, 471.7±124.7µmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MSVAN=0.947×AbbottVAN+0.192 and LC-MSVAN=0.973×AbbottVAN-0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MSVAN=0.866×AbbottVAN+2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration.

Quantification of selected antidepressants and antipsychotics in clinical samples using chromatographic methods combined with mass spectrometry: A review (2006-2015).

BACKGROUND: Psychiatric disorders contribute significantly to worldwide morbidity and mortality. In the case of depression and schizophrenia, effective drug therapy is available but 30-50% of patients do not respond sufficiently to the initial treatment regimen. Apart from the development of new molecules, it is desirable to optimize treatment outcomes with agents that are currently available. Therapeutic drug monitoring (TDM) is a suitable and widely accepted approach for improving the efficacy and safety of these drugs. METHODS: A review of the relevant literature published between 2006 and January 2015. RESULTS AND CONCLUSIONS: This review describes major advances and drawbacks in the field of chromatography coupled with single or tandem mass spectrometry (LC-MS, LC-MS/MS and GC/MS) of selected antidepressants (agomelatine, vilazodone) and antipsychotics (iloperidone, asenapine, amisulpride, aripiprazole, melperone, zotepine, ziprasidone). The high specificity in combination with high sensitivity makes these techniques an attractive complementary method to traditional procedures used in routine practice for TDM.

Fast simultaneous LC/MS/MS determination of 10 active compounds in human serum for therapeutic drug monitoring in psychiatric medication.

A UPLC/MS/MS method with simple protein precipitation has been validated for the fast simultaneous analysis of agomelatine, asenapine, amisulpride, iloperidone, zotepine, melperone, ziprasidone, vilazodone, aripiprazole and its metabolite dehydro-aripiprazole in human serum. Alprenolol was applied as an internal standard. A BEH C18 (2.1 × 50 mm, 1.7 µm) column provided chromatographic separation of analytes using a binary mobile phase gradient (A, 2 mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v; B, 2 mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v). Mass spectrometric detection was performed in the positive electrospray ionization mode and ion suppression owing to matrix effects was evaluated. The validation criteria were determined: linearity, precision, accuracy, recovery, limit of detection, limit of quantification, reproducibility and matrix effect. The concentration range was as follows: 0.25-1000 ng/mL for agomelatine; 0.25-100 ng/mL for asenapine and iloperidone; 2.5-1000 ng/mL for amisulpride, aripiprazole, vilazodone and zotepine; 2.3-924.6 ng/mL for dehydroaripiprazole; 2.2-878.4 ng/mL for melperone; and 2.2-883.5 ng/mL for ziprasidone. Limits of quantitation below a therapeutic reference range were achieved for all analytes. Intra-run precision of 0.4-5.5 %, inter-run precision of 0.6-8.2% and overall recovery of 87.9-114.1% were obtained. The validated method was successfully implemented into routine practice for therapeutic drug monitoring in our hospital.

Liquid chromatography-tandem mass spectrometry method for determination of five antidepressants and four atypical antipsychotics and their main metabolites in human serum.

The rapid and simple ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination parent drugs: sertraline, fluoxetine, citalopram, paroxetine, venlafaxine, clozapine, olanzapine, quetiapine, risperidone, and their active and nonactive metabolites N-desmethylsertraline, norfluoxetine, desmethylcitalopram, didemethylcitalopram, N-desmethylvenlafaxine, O-desmethylvenlafaxine, N-desmethylclozapine, N-desmethylolanzapine, 2-hydroxyolanzapine and 9-hydroxyrisperidone in human serum. Precipitation of serum proteins was performed with a precipitation reagent consisting of 0.05% solution of ZnSO(4)·7H(2)O in acetonitrile/methanol (40:60, v/v). Alprenolol was used as an internal standard. Chromatographic separation was carried out on a BEH C18 column using gradient elution mobile phase A (2 mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v) and B (2 mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v). Electrospray in positive mode was used for ionization. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring. Analysis time was 5 min. Drugs were separated into three groups with low, medium and high levels. Correlation coefficients of calibration curves were in the range 0.995-1.000. Coefficients of variation were 4.2-9.5% for intra-assay and 3.0-11.9% for inter-assay. Recoveries were 87.1-110% for intra-assay and 88.1-108.2% for inter-assay. The method was fully validated and can be successfully applied for routine analyses.