RESUMO
Beta-hydroxybutyric acid (BHB) is a ketone body which is generated from fatty acids as an alternative energy source when glucose is not available. Determination of this compound may be relevant in the forensic laboratory as ketoacidosis - an elevated level of ketone bodies - may contribute to the cause of death. In this study, we aimed at determining the relevance of routinely implementing BHB analysis in the forensic toxicological laboratory, as BHB analysis typically requires an additional workload. We therefore performed an unbiased retrospective analysis of BHB in 599 cases, comprising 553 blood, 232 urine and 62 vitreous humour samples. Cases with BHB concentrations above 100mg/L (in blood, urine and/or vitreous humour) were invariably associated with elevated levels of acetone, another ketone body, the detection of which is already implemented in most forensic laboratories using the gas chromatographic procedure for ethanol quantification. Our retrospective analysis did not reveal any positive case that had been missed initially and confirms that BHB analysis can be limited to acetone positive cases.
Assuntos
Ácido 3-Hidroxibutírico/análise , Acetona/análise , Toxicologia Forense/normas , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Cetose/diagnóstico , Estudos Retrospectivos , Corpo Vítreo/químicaRESUMO
We evaluated the applicability of a validated GC-MS method for the determination of gabapentin in dried blood spots (DBS). Important for the acceptance of DBS sampling as an alternative sampling strategy is the possibility to base solid conclusions on the quantification. Therefore, bridging studies -studies in which the correlation between both DBS and a reference matrix (e.g. serum) is evaluated statistically- need to be conducted. To this end, a comparative study was set up to quantify gabapentin in both blood (DBS) and serum samples. Statistically significant differences between DBS and serum concentrations were found (p<0.001). A mean blood-to-serum ratio of 0.85 was observed, which is in line with expectations. Calculated serum concentrations (obtained by dividing the DBS concentrations by 0.85) demonstrated a good correlation with measured serum concentrations, with 87% of samples fulfilling the criterion for incurred sample reanalysis. Furthermore, our data indicate a good correlation between capillary and venous concentrations. Conclusively, this study demonstrated that DBS are a valid alternative to serum for the determination of gabapentin.
Assuntos
Aminas/análise , Aminas/sangue , Ácidos Cicloexanocarboxílicos/análise , Ácidos Cicloexanocarboxílicos/sangue , Teste em Amostras de Sangue Seco/métodos , Monitoramento de Medicamentos/métodos , Manejo de Espécimes/métodos , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/sangue , Adolescente , Adulto , Cromatografia Líquida de Alta Pressão , Gabapentina , Cromatografia Gasosa-Espectrometria de Massas , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Valores de Referência , Análise de Regressão , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Fatores de Tempo , Adulto JovemRESUMO
Dried blood spot (DBS) sampling and analysis is increasingly being applied in bioanalysis. Although the use of DBS has many advantages, it is also associated with some challenges. E.g. given the limited amount of available material, highly sensitive detection techniques are often required to attain sufficient sensitivity. In gas chromatography coupled to mass spectrometry (GC-MS), derivatization can be helpful to achieve adequate sensitivity. Because this additional sample preparation step is considered as time-consuming, we introduce a new derivatization procedure, i.e. "microwave-assisted on-spot derivatization", to minimize sample preparation of DBS. In this approach the derivatization reagents are directly applied onto the DBS and derivatization takes place in a microwave instead of via conventional heating. In this manuscript we evaluated the applicability of this new concept of derivatization for the determination of two polar low molecular weight molecules, gamma-hydroxybutyric acid (GHB) and gabapentin, in DBS using a standard GC-MS configuration. The method was successfully validated for both compounds, with imprecision and bias values within acceptance criteria (<20% at LLOQ, <15% at 3 other QC levels). Calibration lines were linear over the 10-100µg/mL and 1-30µg/mL range for GHB and gabapentin, respectively. Stability studies revealed no significant decrease of gabapentin and GHB in DBS upon storage at room temperature for at least 84 days. Furthermore, DBS-specific parameters, including hematocrit and volume spotted, were evaluated. As demonstrated by the analysis of GHB and gabapentin positive samples, "microwave-assisted on-spot derivatization" proved to be reliable, fast and applicable in routine toxicology. Moreover, other polar low molecular weight compounds of interest in clinical and/or forensic toxicology, including vigabatrin, beta-hydroxybutyric acid, propylene glycol, diethylene glycol, 1,4-butanediol and 1,2-butanediol, can also be detected using this method.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Micro-Ondas , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/isolamento & purificação , Ácido 3-Hidroxibutírico/normas , Aminas/sangue , Aminas/isolamento & purificação , Aminas/normas , Butileno Glicóis/sangue , Butileno Glicóis/isolamento & purificação , Butileno Glicóis/normas , Calibragem , Ácidos Cicloexanocarboxílicos/sangue , Ácidos Cicloexanocarboxílicos/isolamento & purificação , Ácidos Cicloexanocarboxílicos/normas , Teste em Amostras de Sangue Seco/normas , Toxicologia Forense , Gabapentina , Cromatografia Gasosa-Espectrometria de Massas/normas , Meia-Vida , Humanos , Hidroxibutiratos/sangue , Hidroxibutiratos/isolamento & purificação , Hidroxibutiratos/normas , Peso Molecular , Manejo de Espécimes , Ácido gama-Aminobutírico/sangue , Ácido gama-Aminobutírico/isolamento & purificação , Ácido gama-Aminobutírico/normasRESUMO
BACKGROUND: In Belgium, the analysis of indirect biomarkers such as carbohydrate deficient transferrin (CDT%), gamma-glutamyltransferase (GGT), aspartate aminotransferase/alanine aminotransferase (AST/ALT) and mean corpuscular volume (MCV), is currently used to monitor the alcohol consumption in cases of fitness to drive assessment. We evaluated the use of direct ethanol markers for this purpose, exclusively determined in matrices obtained via non- or minimally invasive sampling. METHODS: Three validated quantitative methods (ethylglucuronide (EtG) in hair and urine, ethylsulfate (EtS) in urine, and phosphatidylethanol species (PEth 16:0/18:1, PEth 18:1/18:1 and PEth 16:0/16:0) in capillary dried blood spots (C-DBS)) were used. Fifty volunteers, for whom fitness to drive had to be assessed and for whom a blood analysis for indirect biomarkers was requested, were included in the study. The sampling and analysis of hair, urine and C-DBS were added to the process currently used. RESULTS: Hair EtG (24/50) and C-DBS PEths (29/50) are more sensitive than the currently used indirect biomarkers (13/50 for CDT%) to detect excessive and chronic alcohol consumption and allow to disprove an abstinence period. Urinary EtG and EtS are useful parameters to determine recent alcohol consumption. CONCLUSION: The combined use of the three strategies allows better inference about the evolution of the alcohol consumption prior to the sampling. Moreover, the exclusive use of non- or minimally invasive sampling (hair, urine and C-DBS) allows this to be performed directly during the fitness to drive assessment by regular staff members. This approach offers the potential to improve the Belgian driver's licence regranting process.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Teste em Amostras de Sangue Seco/normas , Dirigir sob a Influência , Glucuronatos/metabolismo , Cabelo/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , Adulto , Idoso , Consumo de Bebidas Alcoólicas/sangue , Consumo de Bebidas Alcoólicas/urina , Alcoolismo/sangue , Alcoolismo/diagnóstico , Alcoolismo/urina , Bélgica , Biomarcadores/sangue , Biomarcadores/urina , Teste em Amostras de Sangue Seco/métodos , Dirigir sob a Influência/legislação & jurisprudência , Feminino , Glucuronatos/sangue , Glucuronatos/urina , Cabelo/química , Humanos , Masculino , Pessoa de Meia-Idade , Ésteres do Ácido Sulfúrico/sangue , Ésteres do Ácido Sulfúrico/urinaRESUMO
Monitoring of alcohol consumption by living persons takes place in various contexts, amongst which workplace drug testing, driving under the influence of alcohol, driving licence regranting programs, alcohol withdrawal treatment, diagnosis of acute intoxication or fetal alcohol ingestion. The matrices that are mostly used today include blood, breath and urine. The aim of this review is to present alternative sampling strategies that allow monitoring of the alcohol consumption in living subjects. Ethanol itself, indirect (carbohydrate deficient transferrin, CDT%) as well as direct biomarkers (ethyl glucuronide, EtG; ethyl sulphate, EtS; fatty acid ethyl esters, FAEEs and phosphatidylethanol species, PEths) of ethanol consumption will be considered. This review covers dried blood spots (CDT%, EtG/EtS, PEths), dried urine spots (EtG/EtS), sweat and skin surface lipids (ethanol, EtG, FAEEs), oral fluid (ethanol, EtG), exhaled breath (PEths), hair (EtG, FAEEs), nail (EtG), meconium (EtG/EtS, FAEEs), umbilical cord and placenta (EtG/EtS and PEth 16:0/18:1). Main results, issues and considerations specific to each matrix are reported. Details about sample preparation and analytical methods are not within the scope of this review.
Assuntos
Consumo de Bebidas Alcoólicas , Líquidos Corporais , Teste em Amostras de Sangue Seco , HumanosRESUMO
Interindividual variability in the expression and function of drug metabolizing cytochrome P (CYP) 450 enzymes, determined by a combination of genetic, non-genetic and environmental parameters, is a major source of variable drug response. Phenotyping by administration of a selective enzyme substrate, followed by the determination of a specific phenotyping metric, is an appropriate approach to assess the in vivo activity of CYP450 enzymes as it takes into account all influencing factors. A phenotyping protocol should be as simple and convenient as possible. Typically, phenotyping metrics are determined in traditional matrices, such as blood, plasma or urine. Several sampling strategies have been proposed as an alternative for these traditional sampling techniques. In this review, we provide a comprehensive overview of available methods using dried blood spots (DBS), hair, oral fluid, exhaled breath and sweat for in vivo CYP450 phenotyping. We discuss the relation between phenotyping metrics measured in these samples and those in conventional matrices, along with the advantages and limitations of the alternative sampling techniques. Reliable phenotyping procedures for several clinically relevant CYP450 enzymes, including CYP1A2, CYP2C19 and CYP2D6, are currently available for oral fluid, breath or DBS, while additional studies are needed for other CYP450 isoforms, such as CYP3A4. The role of hair analysis for this purpose remains to be established. Being non- or minimally invasive, these sampling strategies provide convenient and patient-friendly alternatives for classical phenotyping procedures, which may contribute to the implementation of CYP450 phenotyping in clinical practice.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Testes Respiratórios , Sistema Enzimático do Citocromo P-450/sangue , Cabelo/enzimologia , Humanos , Fenótipo , Saliva/enzimologia , Manejo de EspécimesRESUMO
Phosphatidylethanol species (PEths) are promising biomarkers of alcohol consumption. Here, we report on the set-up, validation, and application of a novel UHPLC-ESI-MS/MS method for the quantification of PEth 16:0/18:1, PEth 18:1/18:1, and PEth 16:0/16:0 in whole blood (30 µL) and in venous (V, 30 µL) or capillary (C, 3 punches (3 mm)) dried blood spots (DBS). The methods were linear from 10 (LLOQ) to 2000 ng/mL for PEth 16:0/18:1, from 10 (LLOQ) to 1940 ng/mL for PEth 18:1/18:1, and from 19 (LLOQ) to 3872 ng/mL for PEth 16:0/16:0. Extraction efficiencies were higher than 55% (RSD < 18%) and matrix effects compensated for by IS were between 77 and 125% (RSD < 10%). Accuracy, repeatability, and intermediate precision fulfilled acceptance criteria (bias and RSD below 13%). Validity of the procedure for determination of PEth 16:0/18:1 in blood was demonstrated by the successful participation in a proficiency test. The quantification of PEths in C-DBS was not significantly influenced by the hematocrit, punch localization, or spot volume. The stability of PEths in V-DBS stored at room temperature was demonstrated up to 6 months. The method was applied to authentic samples (whole blood, V-DBS, and C-DBS) from 50 inpatients in alcohol withdrawal and 50 control volunteers. Applying a cut-off value to detect inpatients at 221 ng/mL for PEth 16:0/18:1 provided no false positive results and a good sensitivity (86%). Comparison of quantitative results (Bland-Altman plot, Passing-Bablok regression, and Wilcoxon signed rank test) revealed that V-DBS and C-DBS were valid alternatives to venous blood for the detection of alcohol consumption.
Assuntos
Consumo de Bebidas Alcoólicas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Teste em Amostras de Sangue Seco/métodos , Glicerofosfolipídeos/química , Síndrome de Abstinência a Substâncias/sangue , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Biomarcadores/sangue , Biomarcadores/química , Feminino , Glicerofosfolipídeos/sangue , Humanos , Masculino , Pessoa de Meia-Idade , VoluntáriosRESUMO
The degradation and interconversion of a selected set of pterins (dihydroneopterin, hydroxymethyldihydropterin, dihydroxanthopterin, neopterin, hydroxymethylpterin, xanthopterin, 6-formylpterin, 6-carboxypterin and pterin), spiked to charcoal-treated potato and Arabidopsis thaliana matrix was investigated, together with their relative recovery in potato and A. thaliana. As a result, a matrix-specific procedure for the ultra-high performance liquid chromatography-tandem mass spectrometry based determination of 6 aromatic pterins (neopterin, hydroxymethylpterin, xanthopterin, 6-formylpterin, 6-carboxypterin and pterin) is proposed: 1.5ml of an N2-flushed, alkaline (pH=10) extraction solvent is added to 200mg of plant sample. After boiling and homogenization, the samples are incubated: Arabidopsis samples for 30min at room temperature, while shaking, and potato samples for 2h at 37°C (applying a dienzyme treatment with α-amylase and protease). After a final boiling step, the samples are ultrafiltrated and resulting extracts are analyzed by UHPLC-MS/MS.
Assuntos
Arabidopsis/química , Cromatografia Líquida de Alta Pressão/métodos , Extratos Vegetais/química , Pteridinas/química , Solanum tuberosum/química , Espectrometria de Massas em Tandem/métodos , Arabidopsis/metabolismo , Estrutura Molecular , Extratos Vegetais/metabolismo , Pteridinas/metabolismo , Solanum tuberosum/metabolismoRESUMO
A method for the quantitative determination of folates in rodent diets is very important for correct interpretation of folate intake during feeding trials, given the possible discrepancy between the actual folate concentration in the diet and that mentioned on the product sheet. Liquid chromatography tandem-mass spectrometry is the method of choice to differentiate and quantify the individual folate species present. This discrepancy may be accounted for by, e.g., inaccurate folic acid supplementation and/or the presence of endogenous reduced and substituted folates. We developed a method, validated based on FDA guidelines, that allows the measurement of added and endogenous folates by quantitative determination of 5 folate monoglutamates with linear ranges from 8 µg to 2 mg/kg feed. This information, combined with feed intake data, allows insight into the actual folate intake in animal feeding studies. The relevance of this method was illustrated by the analysis of several feed samples of varying composition, by the investigation of the effect of casein incorporation, and by evaluating the variability of the folate content between pellets and production batches.
Assuntos
Ração Animal/análise , Técnicas de Química Analítica/métodos , Ácido Fólico/análogos & derivados , Glutamatos/análise , Animais , Ácido Fólico/análise , RoedoresRESUMO
Caffeine is the probe drug of choice to assess the phenotype of the drug metabolizing enzyme CYP1A2. Typically, molar concentration ratios of paraxanthine, caffeine's major metabolite, to its precursor are determined in plasma following administration of a caffeine test dose. The aim of this study was to develop and validate an LC-MS/MS method for the determination of caffeine and paraxanthine in hair. The different steps of a hair extraction procedure were thoroughly optimized. Following a three-step decontamination procedure, caffeine and paraxanthine were extracted from 20 mg of ground hair using a solution of protease type VIII in Tris buffer (pH 7.5). Resulting hair extracts were cleaned up on Strata-X™ SPE cartridges. All samples were analyzed on a Waters Acquity UPLC® system coupled to an AB SCIEX API 4000™ triple quadrupole mass spectrometer. The final method was fully validated based on international guidelines. Linear calibration lines for caffeine and paraxanthine ranged from 20 to 500 pg/mg. Precision (%RSD) and accuracy (%bias) were below 12% and 7%, respectively. The isotopically labeled internal standards compensated for the ion suppression observed for both compounds. Relative matrix effects were below 15%RSD. The recovery of the sample preparation procedure was high (>85%) and reproducible. Caffeine and paraxanthine were stable in hair for at least 644 days. The effect of the hair decontamination procedure was evaluated as well. Finally, the applicability of the developed procedure was demonstrated by determining caffeine and paraxanthine concentrations in hair samples of ten healthy volunteers. The optimized and validated method for determination of caffeine and paraxanthine in hair proved to be reliable and may serve to evaluate the potential of hair analysis for CYP1A2 phenotyping.
Assuntos
Cafeína/análise , Cafeína/isolamento & purificação , Cromatografia Líquida/métodos , Cabelo/química , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Teofilina/análise , Teofilina/isolamento & purificação , Adulto , Métodos Analíticos de Preparação de Amostras , Cafeína/metabolismo , Feminino , Humanos , Masculino , Teofilina/metabolismo , Adulto JovemRESUMO
AIM: DBS sampling has been proposed as an alternative for venous blood collection in therapeutic drug monitoring (TDM) of antipsychotics. For implementation in routine practice, a comparison between capillary and venous blood concentrations is mandatory. RESULTS: A DBS method for quantification of antipsychotics was clinically validated. First, whole blood therapeutic ranges were calculated using the blood:serum ratio. Calculation of DBS:blood ratios and Passing-Bablok regression analysis demonstrated that concentrations obtained by DBS analysis were highly comparable to those obtained by conventional whole blood analysis. Clinical interpretation of serum, whole blood and DBS concentrations were highly identical (sensitivity 91.6-97.6%). CONCLUSION: This is the first clinical study demonstrating the value of DBS sampling in TDM of antipsychotics.
Assuntos
Antipsicóticos/sangue , Teste em Amostras de Sangue Seco/métodos , Monitoramento de Medicamentos/métodos , Administração Oral , Adulto , Idoso , Antipsicóticos/administração & dosagem , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Soro , Adulto JovemRESUMO
Volumetric absorptive microsampling (VAMS) is a novel sampling technique that allows the straightforward collection of an accurate volume of blood (approximately 10µL) from a drop or pool of blood by dipping an absorbent polymeric tip into it. The resulting blood microsample is dried and analyzed as a whole. The aim of this study was to evaluate the potential of VAMS to overcome the hematocrit bias, an important issue in the analysis of dried blood microsamples. An LC-MS/MS method for analysis of the model compounds caffeine and paraxanthine in VAMS samples was fully validated and fulfilled all pre-established criteria. In conjunction with previously validated procedures for dried blood spots (DBS) and blood, this allowed us to set up a meticulous comparative study in which both compounds were determined in over 80 corresponding VAMS, DBS and liquid whole blood samples. These originated from authentic human patient samples, covering a wide hematocrit range (0.21-0.50). By calculating the differences with reference whole blood concentrations, we found that analyte concentrations in VAMS samples were not affected by a bias that changed over the evaluated hematocrit range, in contrast to DBS results. However, VAMS concentrations tend to overestimate whole blood concentrations, as a consistent positive bias was observed. A different behavior of VAMS samples prepared from incurred and spiked blood, combined with a somewhat reduced recovery of caffeine and paraxanthine from VAMS tips at high hematocrit values, an effect that was not observed for DBS using a very similar extraction procedure, was found to be at the basis of the observed VAMS-whole blood deviations. Based on this study, being the first in which the validity and robustness of VAMS is evaluated by analyzing incurred human samples, it can be concluded that VAMS effectively assists in eliminating the effect of hematocrit.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Cafeína/sangue , Teste em Amostras de Sangue Seco/métodos , Teofilina/sangue , Cromatografia Gasosa , Voluntários Saudáveis , Hematócrito , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
A stable isotope dilution LC-MS/MS method is the method of choice for the selective quantitative determination of several folate species in clinical samples. By implementing an integrated approach to determine both the plasma and red blood cell (RBC) folate status, the use of consumables and time remains limited. Starting from a single 300µl whole blood sample, the folate status in plasma and RBCs can be determined after separating plasma and RBCs and sequential washing of the latter with isotonic buffer, followed by reproducible lysis using an ammonium-based buffer. Acidification combines both liberation of protein bound folates and protein precipitation. Sample cleanup is performed using a 96-well reversed-phase solid-phase extraction procedure, similar for both plasma and RBC samples. Analyses are performed by UHPLC-MS/MS. Method validation was successfully performed based on EMA-guidelines and encompassed selectivity, carry-over, linearity, accuracy, precision, recovery, matrix effect and stability. Plasma and RBC folates could be quantified in the range of 1-150nmol/l and 5-1500nmol/l, respectively. This method allows for the determination of 6 folate monoglutamates in both plasma and RBCs. It can be used to determine short and long term folate status in both normal and severely deficient subjects in a single analytical sequence.
Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Eritrócitos/química , Ácido Fólico/análise , Ácido Fólico/sangue , Plasma/química , Espectrometria de Massas em Tandem , Ácido Fólico/análogos & derivados , Glutamatos/análise , HumanosRESUMO
BACKGROUND AND OBJECTIVE: Although metabolite-to-parent drug concentration ratios in hair have been suggested as a possible tool to study the metabolism of drugs in a non-invasive way, no studies are available that evaluated this in a systematic way. Cytochrome P450 (CYP) 1A2 is a drug-metabolizing enzyme characterized by large inter-individual differences in its activity. The standard approach for CYP1A2 phenotyping is to determine the paraxanthine/caffeine ratio in plasma at a fixed timepoint after intake of a dose of the CYP1A2 substrate caffeine. The aim of this study was to evaluate whether paraxanthine/caffeine ratios measured in hair samples reflect the plasma-based CYP1A2 phenotype. METHODS: Caffeine and paraxanthine concentrations were measured in proximal 3 cm segments of hair samples from 60 healthy volunteers and resulting paraxanthine/caffeine ratios were correlated with CYP1A2 phenotyping indices in plasma. RESULTS: Paraxanthine/caffeine ratios in hair ranged from 0.12 to 0.93 (median 0.41); corresponding ratios in plasma ranged from 0.09 to 0.95 (median 0.40). A statistically significant correlation was found between ratios in hair and plasma (r = 0.41, p = 0.0011). However, large deviations between ratios in both matrices were found in individual cases. Although the influence of several factors on paraxanthine/caffeine ratios and hair-plasma deviations was investigated, no evident factors explaining the observed variability could be identified. CONCLUSION: The variability between hair and plasma ratios complicates the interpretation of hair paraxanthine/caffeine ratios on an individual basis and, therefore, compromises their practical usefulness as alternative CYP1A2 phenotyping matrix.
Assuntos
Cafeína/farmacocinética , Citocromo P-450 CYP1A2/metabolismo , Cabelo/metabolismo , Teofilina/farmacocinética , Adulto , Cafeína/análise , Cafeína/sangue , Citocromo P-450 CYP1A2/sangue , Feminino , Cabelo/química , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Teofilina/análise , Teofilina/sangue , Adulto JovemRESUMO
Gamma-hydroxybutyric acid (GHB) is a well-known illicit club and date-rape drug. Dried blood spot (DBS) sampling is a promising alternative for classical venous sampling in cases of (suspected) GHB intoxication since it allows rapid sampling, which is of interest for the extensively metabolized GHB. However, there is limited data if -and how- capillary DBS concentrations correlate with venous concentrations. We conducted a comparative study in 50 patients with suspected GHB intoxication, to determine and to correlate GHB concentrations in venous DBS (vDBS) and capillary DBS (cDBS). This is the first study that evaluates in a large cohort the correlation between capillary and venous concentrations of an illicit drug in real-life samples. Of the 50 paired samples, 7 were excluded: the vDBS concentration was below the LLOQ of 2 µg/mL in 3 cases and 4 samples were excluded after visual inspection of the DBS. Bland-Altman analysis revealed a mean % difference of -2.8% between cDBS and vDBS concentrations, with the zero value included in the 95% confidence interval of the mean difference in GHB concentration. A paired sample t-test confirmed this observation (p = 0.17). Also the requirement for incurred sample reproducibility was fulfilled: for more than two-thirds of the samples the concentrations obtained in cDBS and those in vDBS were within 20% of their mean. Since equivalent concentrations were observed in cDBS and vDBS, blood obtained by fingerprick can be considered a valid alternative for venous blood for GHB determination.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Hidroxibutiratos/sangue , Humanos , Reprodutibilidade dos TestesRESUMO
Therapeutic drug monitoring of antipsychotics is important in optimizing individual therapy. In psychiatric populations, classical venous blood sampling is experienced as frightening. Interest in alternative techniques, like dried blood spots (DBS), has consequently increased. A fast and easy to perform DBS method for quantification of 16 antipsychotics (amisulpride, aripiprazole, asenapine, bromperidol, clozapine, haloperidol, iloperidone, levosulpiride, lurasidone, olanzapine, paliperidone, pipamperone, quetiapine, risperidone, sertindole and zuclopenthixol) and 8 metabolites was developed. DBS were prepared using 25 µL of whole blood and extraction of complete spots was performed using methanol: methyl-t-butyl-ether (4:1). After evaporation, the extract was reconstituted in the mobile phase and 10 µL were injected on an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Separation using a C18 column and gradient elution with a flow rate of 0.5 mL/min resulted in a 6-min run-time. Ionization was performed in positive mode and a dynamic MRM method was applied. Median recovery was 66.4 % (range 28.7-84.5%). Accuracy was within the acceptance criteria, except for pipamperone (LLOQ and low concentration) and lurasidone (low concentration). Imprecision was only aberrant for lurasidone at low and medium concentration. All compounds were stable during 1 month at room temperature, 4 °C and -18 °C. Lurasidone was unstable when the extract was stored for 12 h on the autosampler. Absolute matrix effects (ME) (median 66.1%) were compensated by the use of deuterated IS (median 98.8%). The DBS method was successfully applied on 25-µL capillary DBS from patients and proved to be a reliable alternative for quantification of all antipsychotics except for olanzapine and N-desmethylolanzapine.
Assuntos
Antipsicóticos/sangue , Teste em Amostras de Sangue Seco , Monitoramento de Medicamentos/métodos , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas em TandemRESUMO
The Society of Hair Testing (SoHT) has provided cutoffs for the quantification of ethyl glucuronide (EtG) in hair to indicate occasional or chronic/excessive alcohol consumption. Although several sensitive methods have been reported, past proficiency test results show a lack of reproducibility. An ultra-performance liquid chromatographic mass spectrometric method (LLOQ of 10 pg EtG/mg hair) has been validated according to the international guidelines, including the successful participation in five proficiency tests. This method was subsequently used to evaluate the impact of different grinding conditions (cut, weakly or extensively pulverized hair samples) on the final measured EtG concentration. Hair from alcohol consumers (n = 2) and commercially available quality control samples (QCs) (n = 2) was used. For the QCs, extensive pulverization led to a significantly higher amount of measured EtG. In the hair samples obtained from volunteers, cut or weakly pulverized hair resulted in EtG concentrations below the LLOQ, while the mean concentrations of 14 and 40 pg EtG/mg hair were determined after extensive pulverization. Differences in sample preparation could partially explain inter-laboratory variability. As the differences in results can lead to a different interpretation even when applying the SoHT cutoffs, it is of interest to standardize sample preparation techniques in the field of EtG hair testing.
Assuntos
Glucuronatos/análise , Cabelo/química , Manejo de Espécimes , Consumo de Bebidas Alcoólicas , Alcoolismo/diagnóstico , Cromatografia Líquida , Feminino , Humanos , Masculino , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
SCOPE: The biological impact of folates from folate rice, a metabolically engineered (biofortified) rice line, rich in folates, was investigated. Its consumption may be helpful to fight folate deficiency. Our objective was to investigate the potential of folate rice to supply the organism with folates and evaluate its biological effectiveness using a rat model. METHODS AND RESULTS: Five groups of 12 Wistar rats were monitored during a 7/12-wk depletion/repletion trial. Animals receiving folate-free diet (0 µg/rat/day) and those additionally receiving wild-type rice (on average 0.11 µg/rat/day) suffered from decreased hematocrit and lower folate concentrations in both plasma and RBCs. This resulted in serious morbidity and even lethality during the trial. In contrast, all animals receiving a daily supplement of folate rice or folic acid fortified rice (on average 3.00 µg/rat/day and 3.12 µg/rat/day, respectively) and those receiving a positive control diet (11.4 to 25.0 µg/rat/day), survived. In these groups, the hematocrit normalized, plasma and RBC folate concentrations increased and pronounced hyperhomocysteinemia was countered. CONCLUSION: Using an animal model, we demonstrated that biofortified folate rice is a valuable source of dietary folate, as evidenced by folate determination in plasma and RBCs, the alleviation of anemia and counteraction of pronounced hyperhomocysteinemia.
Assuntos
Ácido Fólico/farmacologia , Oryza/química , Plantas Geneticamente Modificadas/química , Animais , Peso Corporal/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Ácido Fólico/sangue , Alimentos Fortificados , Hematócrito , Homocisteína/sangue , Estudos Longitudinais , Oryza/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ratos Wistar , Vitamina B 12/sangueRESUMO
Dried blood spot (DBS) sampling and DBS analysis have increasingly received attention during recent years. Furthermore, a substantial number of DBS methods has recently become available in clinical, forensic and occupational toxicology. In this review, we provide an overview of the different DBS-based methods that have been developed for detecting (markers of) abused substances. These include both legal and illegal drugs belonging to different categories, including cannabinoids, cocaine and metabolites, opioids, benzodiazepines and Z-drugs, amphetamines and analogs, gamma-hydroxybutyric acid, ketamine and novel psychoactive substances such as cathinones. Markers of ethanol consumption and tobacco use are also covered in this review. Since the majority of published methods has shown promising results overall, an interesting role for DBS analysis in diverse toxicological applications can be envisaged. For the distinct applications, we discuss the specific potential and benefits of DBS, the associated limitations and challenges, as well as recent developments and future perspectives.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Detecção do Abuso de Substâncias/métodos , Animais , Estabilidade de Medicamentos , Toxicologia Forense , Humanos , Limite de DetecçãoRESUMO
BACKGROUND: Phenotyping, using caffeine as probe substrate, is a proper method to assess CYP1A2 activity. We evaluated the utility of dried blood spots (DBS) for CYP1A2 phenotyping. RESULTS: LC-MS/MS methods were developed and validated for quantitation of caffeine and its metabolite paraxanthine in DBS, whole blood and plasma. All parameters met the pre-established criteria. While recovery, matrix effects and precision were unaffected by hematocrit (Hct), there was a Hct effect on accuracy, although for the evaluated Hct interval (0.36-0.50) it remained within acceptable limits. The phenotyping methods were successfully applied in healthy volunteers. CONCLUSION: Excellent method performance and highly comparable phenotyping indices in DBS, whole blood and plasma, combined with the benefits of DBS sampling, illustrate the suitability of DBS-based CYP1A2 phenotyping.