Stability of Lisdexamfetamine in Sampled Whole Blood-Implications of Sampling Tube Additives and Storage Temperature for Interpretation of Impairment.

Lisdexamfetamine (LDX) is a prodrug that is enzymatically converted into dextroamphetamine (d-AMP), a central nervous system stimulant. The stability of LDX in sampled whole blood is an important issue that may be crucial in the assessment of impaired driving caused by d-AMP. This study investigated the stability of LDX in whole blood collected in two different tubes containing a fluoride oxalate (FX) mixture and a fluoride citrate (FC) mixture. Without additives, LDX was unstable. LDX was also unstable in FX blood stored at ambient temperature or 4°C. After 3 days of storage at ambient temperature, an initial LDX concentration of 47 ± 1 ng/g (mean ± SD) was no longer detectable in the samples (n = 3). Instead, 19 ± 0.6 ng/g d-AMP was formed. The stability was improved at 4°C. After 7 days of storage at 4°C, 88 ± 5% of an initial LDX concentration of 50 ± 0.4 ng/g was recovered and 3.8 ± 0.3 ng/g d-AMP was formed. The stability of LDX was greater in FC blood than in FX blood; 79 ± 10% and 93 ± 4% of initial LDX concentrations of 48 ± 2 and 51 ± 0.5 ng/g were recovered from FC blood after 7 days of storage at ambient temperature and 4°C, respectively, and the corresponding formation of d-AMP was 5.8 ± 0.6 and 0.5 ± 0.3 ng/g, respectively. When FX and FC blood were stored at -20°C or -80°C, no detectable degradation of LDX or formation of d-AMP was observed after 3 weeks of storage.

Senicapoc treatment in COVID-19 patients with severe respiratory insufficiency-A randomized, open-label, phase II trial.

BACKGROUND: The aim of the current study was to determine if treatment with senicapoc, improves the PaO2 /FiO2 ratio in patients with COVID-19 and severe respiratory insufficiency. METHODS: Investigator-initiated, randomized, open-label, phase II trial in four intensive care units (ICU) in Denmark. We included patients aged ≥18 years and admitted to an ICU with severe respiratory insufficiency due to COVID-19. The intervention consisted of 50 mg enteral senicapoc administered as soon as possible after randomization and again after 24 h. Patients in the control group received standard care only. The primary outcome was the PaO2 /FiO2 ratio at 72 h. RESULTS: Twenty patients were randomized to senicapoc and 26 patients to standard care. Important differences existed in patient characteristics at baseline, including more patients being on non-invasive/invasive ventilation in the control group (54% vs. 35%). The median senicapoc concentration at 72 h was 62.1 ng/ml (IQR 46.7-71.2). The primary outcome, PaO2 /FiO2 ratio at 72 h, was significantly lower in the senicapoc group (mean 19.5 kPa, SD 6.6) than in the control group (mean 24.4 kPa, SD 9.2) (mean difference -5.1 kPa [95% CI -10.2, -0.04] p = .05). The 28-day mortality in the senicapoc group was 2/20 (10%) compared with 6/26 (23%) in the control group (OR 0.36 95% CI 0.06-2.07, p = .26). CONCLUSIONS: Treatment with senicapoc resulted in a significantly lower PaO2 /FiO2 ratio at 72 h with no differences for other outcomes.

The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

BACKGROUND: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. METHODS: We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. FINDINGS: We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. INTERPRETATION: Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. FUNDING: NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.

A validated UHPLC-MS/MS method for rapid determination of senicapoc in plasma samples.

The clinically tested KCa3.1 channel blocker, senicapoc, has been proven to have excellent pharmacological properties and prior clinical trials found it to be safe for use in patients with sickle cell anaemia. Currently, several preclinical projects are aiming to repurpose senicapoc for other indications, but well-described analytical methods in the literature are lacking. Our aim was to develop a sensitive, rapid and accurate ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) suitable for the determination of senicapoc in plasma samples. Unfortunately, direct analysis of senicapoc in crude acetonitrile extracts of human plasma samples by UHPLC-ESI-MS/MS was subjected to significant and variable ion suppression from coeluting phospholipids (PLs). The interferences were mainly caused by the presence of phosphatidylcholine and phosphatidylethanolamine classes of PLs, including their lyso-products. However, the PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties which decreased the total ion suppression effect to approximately 5%. Based on this technique, a simple high-throughput UHPLC-MS/MS method was developed and validated for the determination of senicapoc in 100-µL plasma samples. The lower limit of quantification was 0.1 ng/mL. The mean true extraction recovery was close to 100 %. The relative intra-laboratory reproducibility standard deviations of the measured concentrations were 8% and 4% at concentrations of 0.1 ng/mL and 250 ng/mL, respectively. The trueness expressed as the relative bias of the test results was within ± 2% at concentrations of 1 ng/mL or higher.

Entrapment of drugs in dental calculus - Detection validation based on test results from post-mortem investigations.

For prospective investigation of drugs and metabolites in archaeological and contemporary dental calculus, a sensitive, broadly applicable ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) was developed. The dental calculus was treated with citric acid and the dissolution extracts were cleaned using weak and strong polymeric cation-exchange sorbents. The method was validated on hydroxyapatite for the analysis of 67 drugs and metabolites. Typically, the lower limits of quantification were in the range of 0.01-0.05ng for the sample mass extracted. The general applicability of the method was tested using dental calculus material sampled from 10 corpses undergoing forensic autopsy. The calculus material was washed several times before dissolution to remove residual substances originating from saliva, gingival crevicular fluid and blood. The wash extracts and the calculus samples (cleaned calculus material) were analysed using the same instrumental conditions. The dry mass of the calculus samples ranged from 1 to 10mg. The total number of drug detections was 131 in the dental calculus samples and 117 in the whole blood samples. From the analyses of the wash extracts and calculus samples, it was proven that drug residues were trapped in the interior of the calculus material. In 82 of the drug detections, the drug concentrations were higher in the dental calculus than in the blood. Among substances detected in the dental calculus but not in the blood were cocaine, heroin, 6-MAM and THCA-A.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.

Ascorbic Acid Improves the Stability of Buprenorphine in Frozen Whole Blood Samples.

Buprenorphine (BUP) was not long-term stable in whole blood samples preserved with fluoride citrate (FC) and fluoride oxalate (FX) mixtures when stored at -20°C. On average, only half of the initial concentrations of BUP was recovered after 12 weeks of storage at -20°C when interrupted by 3-4 thaw/freeze cycles. Norbuprenorphine (NBUP) was less unstable; approximately 90% was recovered after 12 weeks of storage at -20°C. The instability was less at 5°C, but the formation of BUP and NBUP from their glucuronides was observed at that temperature, especially in FC-preserved blood. The substances were stable for at least 5 months when stored uninterrupted at -80°C. The instability of BUP and NBUP in FC- and FX-preserved whole blood stored at -20°C was eliminated when the samples were modified with 30 mM ascorbic acid (ASC) prior to storage. The mean recoveries were greater than 95% after a 5-month interrupted storage period at -20°C when modified with ASC.

Sensitive determination of monoamine neurotransmitters, their main metabolites and precursor amino acids in different mouse brain components by liquid chromatography-electrospray tandem mass spectrometry after selective sample clean-up.

For the assessment of diets and supplements formulated for the treatment of phenylketonuria, a highly sensitive and selective method was developed and validated for the quantification of dopamine (DA), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), phenylalanine, tyrosine and tryptophan in mouse cerebellum, brain stem, hypothalamus, parietal cortex, anterior piriform cortex and bulbus olfactorius. Samples were extracted by deproteinization with acetonitrile, and the extracts were cleaned up by strong anion exchange and weak cation exchange applied sequentially. The substances were detected by rapid liquid chromatography tandem mass spectrometry. Matrix components were largely removed by the clean-up, resulting in low matrix effects. The lower limits of quantification for an extracted tissue mass of 100 mg were 0.3, 0.3, 0.2 and 2 ng/g for DA, 5-HT, 5-HIAA and DOPAC, respectively. The mean true extraction recoveries were 80-102%. The relative intra-laboratory reproducibility standard deviations were generally <11% at concentrations of 20-1000 ng/g for DA, 5-HT, 5-HIAA and DOPAC and 7% at concentrations of 5-50 µg/g for the amino acids. This method was successfully used in a phenylketonuria mice study including nearly 300 brain tissue samples and for small sample masses (for example, 2 mg of bulbus olfactorius).

The effect of antioxidants on the long-term stability of THC and related cannabinoids in sampled whole blood.

The stability of cannabinoids is complex and crucial for the assessment of impaired driving caused by cannabis. Therefore, the effect of antioxidants on the long-term stability of Δ9 -tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-Δ9 -tetrahydrocannabinol (THC-OH), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in whole blood samples preserved with fluoride citrate (FC) and fluoride oxalate (FX) mixtures was investigated at different temperatures. The measured concentrations of the cannabinoids in authentic whole blood preserved solely with FC or FX mixtures decreased significantly during prolonged storage at -20°C. On average, less than 5% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage interrupted by 5 thawing/freezing cycles. The rate of decrease was greatest in FC-preserved blood. The repeated thawing/freezing of the samples accelerated the instability progression. At 5°C approximately 60% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage. No significant decrease was observed in samples stored at -80°C during the test period of 5 months. The instability at -20°C was to a great extend avoided by adding 30 mM ascorbic acid (ASC) to the samples before storage. Samples preserved with a combination of the FX mixture and ASC showed no significant decrease in the recovered concentrations during a 5-month storage period interrupted by 6 thawing/freezing cycles. Samples preserved with a combination of the FC mixture and ASC showed almost similar improvements in cannabinoid stability. Other reducing agents such as sodium metabisulfite and glutathione also improved the stability in FX-preserved blood stored at -20°C.

cGAS is activated by DNA in a length-dependent manner.

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length-dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.

Sensitive Determination of Cannabinoids in Whole Blood by LC-MS-MS After Rapid Removal of Phospholipids by Filtration.

Direct analysis of Δ9-tetrahydrocannabinol (THC) and other cannabinoids in crude acetonitrile extracts of whole blood by liquid chromatography-tandem mass spectrometry using pneumatically assisted electrospray ionization (LC-ESI-MS-MS) was subjected to pronounced ion suppression from co-eluting phospholipids (PLs). The interferences were mainly caused by the lysophosphatidylcholine and lysophosphatidylethanolamine classes of PLs. The PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties, which typically increased the THC and cannabinol (CBN) signal intensities by a factor of 5. Based on this technique, a simple high-throughput LC-MS-MS method was developed for the determination of cannabinoids in 100 µL samples of whole blood. The lower limits of quantification were 0.2 µg/L for THC, CBN, cannabidiol (CBD) and Δ9-tetrahydrocannabinolic acid A (THCA-A) and 0.5 µg/L for 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). The mean ion suppression levels after clean-up were 10% (THC), 9% (CBN), 17% (CBD), 0% (THC-OH), 2% (THC-COOH) and 9% (THCA-A) at blood concentration levels of 1-10 µg/L. The mean true extraction recoveries were 97% (THC), 101% (CBN), 101% (CBD), 98% (THC-OH), 95% (THC-COOH) and 90% (THCA-A) at the same concentration levels. The relative intra-laboratory reproducibility standard deviations were <9% at concentrations of 1 µg/L or higher. The trueness expressed as the relative bias of the test results was within ±4% at concentrations of 1 µg/L or higher.

Simultaneous determination of propofol and its glucuronide in whole blood by liquid chromatography-electrospray tandem mass spectrometry and the influence of sample storage conditions on the reliability of the test results.

Propofol (2,6-diisopropylphenol) is commonly used as an anaesthetic agent but is also abused for recreational purposes. Several cases of fatalities involving self-administered propofol have been reported. For rapid quantification of propofol and propofol ß-d-glucuronide (propofol G) in clinical and forensic cases, an ultra-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation has been developed. The technique has been validated on both ante-mortem and post-mortem human whole blood. The proteins in the blood samples were removed by the addition of a mixture of methanol and acetonitrile, and the extract was cleaned up by solid phase extraction. The extract was concentrated in dimethyl sulphoxide. The system was calibrated using matrix-matched calibrants combined with isotope dilution. The lower limits of quantification were 0.01 and 0.02mg/L for propofol and 0.02 and 0.04mg/L for propofol G in ante-mortem and post-mortem whole blood, respectively. The relative intra-laboratory reproducibility standard deviation was less than 10% at concentrations of 0.2mg/L or higher. The mean true extraction recovery was 85% for propofol and 81% for propofol G. The trueness of the propofol determination expressed as the relative bias of the test results was within ±6% at concentration levels of 0.01-8.5mg/L. Propofol was less stable in blood stabilised with a citrate-EDTA-fluoride mixture than in blood stabilised with an oxalate-fluoride mixture. The stability was lower at -20°C than at 5°C and -80°C. Propofol G did not show instability under the storage conditions tested.

Determination of therapeutic γ-aminobutyric acid analogs in forensic whole blood by hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry.

Vigabatrin, pregabalin, gabapentin and baclofen are γ-aminobutyric acid analogs that are used in the treatment of epileptic seizures (vigabatrin, pregabalin and gabapentin) and spasticity (baclofen). The intake of these drugs may induce adverse reactions and impair the ability of an individual to drive a vehicle. There have also been reports of cases of intoxication and fatalities from overdoses. For rapid and accurate quantification of these drugs in forensic cases, an ultraperformance liquid chromatography tandem mass spectrometry method using pneumatically assisted electrospray ionization has been developed. The technique has been validated on both ante- and postmortem human whole blood. The protein in the blood samples was removed by the addition of a mixture of methanol and acetonitrile, and the extract was ultrafiltered and diluted with acetonitrile. The separation was performed by hydrophilic interaction liquid chromatography. Calibration of the system was achieved through use of matrix-matched calibrants combined with isotope dilution. The lower limits of quantification were 0.02-0.04 mg/L, and the relative intra-laboratory reproducibility standard deviations were <4 and 8% at concentrations of 10 and 1 mg/L, respectively. The mean true recoveries were >89%. The trueness expressed as the relative bias of the test results was within ±7% at concentrations of 1-40 mg/L for vigabatrin, pregabalin and gabapentin and of 0.1-4 mg/L for baclofen.

Simultaneous determination of ß-hydroxybutyrate and ß-hydroxy-ß-methylbutyrate in human whole blood using hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry.

OBJECTIVES: For the quantification of ß-hydroxybutyrate (BHB) and ß-hydroxy-ß-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was developed, which does not require chemical modification of the analytes. DESIGN AND METHODS: Samples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis. RESULTS: Separation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10-20µM BHB and 1µM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ±5% at concentration levels of 10-1000µM BHB and 1-20µM HMB. The lower limits of quantification were estimated to be 3µM for BHB and 0.4µM for HMB. CONCLUSIONS: A simple and highly sensitive and selective HILIC-MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood.

A hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry method for the simultaneous determination of γ-hydroxybutyrate and its precursors in forensic whole blood.

A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of γ-hydroxybutyric acid (GHB), γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in human ante-mortem and post-mortem whole blood. The blood proteins were precipitated using a mixture of methanol and acetonitrile, and the extract was cleaned-up by passage through a polymeric strong cation exchange sorbent. Separation of the analytes and their structural isomers was obtained using a column with a zwitterionic stationary phase. Matrix-matched calibrants, combined with isotope dilution, were used for quantitative analysis. GHB was determined in both positive and negative ion modes. The relative intra-laboratory reproducibility standard deviations were better than 10% and 6% for blood samples at concentrations of 2 mg/L and 20-150 mg/L, respectively. The mean true extraction recoveries were 80% for GHB and greater than 90% for GBL and 1,4-BD at concentration levels of 20-50 mg/L. The limits of detection were approximately 0.5 mg/L for GHB and GBL, and 0.02 mg/L for 1,4-BD in ante-mortem blood. The corresponding lower limits of quantification were less than 1 mg/L for GHB and GBL, and less than 0.1 mg/L for 1,4-BD. GBL was unstable in whole blood freshly preserved with a sodium fluoride oxalate mixture, but the stability could be improved significantly by preservation with a sodium fluoride citrate EDTA mixture.

A liquid chromatography-electrospray tandem mass spectrometry method for the determination of antiarrhythmic drugs and their metabolites in forensic whole blood samples.

A liquid chromatography-tandem mass spectrometry method, using pneumatically assisted electrospray ionization was developed for the determination of amiodarone, desethylamiodarone, propafenone, N-depropylpropafenone, 5-OH-propafenone, flecainide, and sotalol in human antemortem and postmortem whole blood. A mixture of methanol and acetonitrile was used to extract the samples, and the clear extracts obtained from the protein precipitation were injected directly onto an ethyl-linked phenyl LC column. The isotope dilution technique was applied for quantitative analysis. The relative intralaboratory reproducibility standard deviations were 5-9% at a concentration range of 1-5 mg/L and 9-12% at a concentration of 0.1 mg/L. The mean true recoveries were greater than 91% in the concentration range of 0.05-5 mg/L. The detection limits were in the range of 8-18 µg/L.

Determination of metformin and other biguanides in forensic whole blood samples by hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry.

Metformin is an anti-diabetic drug in the biguanide class which also includes phenformin and buformin. Because of the potential adverse effects of the biguanides, a reliable liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionization was developed for the quantification of the drugs in both live and post-mortem human whole blood. The blood proteins were precipitated by the addition of a mixture of methanol and acetonitrile, and the extract was cleaned up by cation-exchange solid-phase extraction to eliminate ion suppression effects. The separation was performed by hydrophilic interaction liquid chromatography. Matrix-matched calibrants combined with isotope dilution of metformin were used for calibration. The detection limits were 0.01 mg/L for metformin and phenformin and the relative intra-laboratory reproducibility standard deviations were less than 6% at concentrations of 1-10 mg/L. The mean true recoveries were greater than 86%.

Determination of cathinones and related ephedrines in forensic whole-blood samples by liquid-chromatography-electrospray tandem mass spectrometry.

A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of cathinone, methcathinone, ethcathinone, amfepramone, mephedrone, flephedrone, methedrone, methylone, butylone, cathine, norephedrine, ephedrine, pseudoephedrine, methylephedrine and methylpseudoephedrine in human live and post-mortem whole blood. The blood proteins were precipitated by the addition of methanol, and the extract was purified by ultrafiltration. The separation of diastereomeric ephedrines was achieved on an ethyl-linked phenyl column. Matrix-matched calibrants combined with the isotope dilution of selected substances were used for quantitative analysis. The relative intra-laboratory reproducibility standard deviations were generally better than 7% at concentrations of 20 µg/L, and the mean true recoveries were 87-106% in the concentration range of 10-250 µg/L. The detection limits were in the range of 0.5-3 µg/L. The cathinones were unstable in whole blood and sample extracts under neutral conditions, but the stability could be improved by the acidification of the sample matrix.