Evaluation of a candidate reference measurement procedure for serum free testosterone based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry.

BACKGROUND: To assess the analytical validity of free testosterone (FTe) measurements, a reference measurement procedure (RMP) is required. For steroids, isotope dilution-mass spectrometry is accepted as state-of-the-art technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the protein-bound fraction, the RMP should include a physical separation step. The use of equilibrium dialysis (ED) or ultrafiltration (UF) is advocated. Our objective was to develop such a candidate RMP. METHODS: We selected UF combined with isotope dilution-gas chromatography-mass spectrometry (ID-GC/MS) for direct measurement of Te in the ultrafiltrate. After optimization of the UF process, the complete procedure was validated by use of split-sample comparisons with indirect ED (iED) and symmetric dialysis (SyD). RESULTS: The candidate RMP gave maximum within-day, between-day, and total CVs of 3.0%, 3.1%, and 4.3%. The Deming regression equations for the respective method comparisons were: UF-ID-GC/MS = 0.98(iED) - 53 pmol/L (r = 0.94; S(y|x)= 42 pmol/L) and UF-ID-GC/MS = 0.92(SyD) + 21 pmol/L (r = 0.97; S(y|x)= 31 pmol/L). CONCLUSIONS: We achieved the objective of a state-of-the-art candidate RMP, which agreed well with iED and SyD. However, we also demonstrated that a degree of discordance remains, which may require a decision from an authoritative organization on the recommended procedure to measure free hormone concentrations.

Application of a C-peptide electrospray ionization-isotope dilution-liquid chromatography-tandem mass spectrometry measurement procedure for the evaluation of five C-peptide immunoassays for urine.

This study applied electrospray ionization-isotope dilution-liquid chromatography-tandem mass spectrometry for the evaluation of five urinary C-peptide immunoassays via split-sample measurements. The immunoassays measured in duplicate in the same run, the comparison method in triplicate over different runs. From the data, the within-run imprecision and the method comparison total RSDs were calculated. Regression analysis revealed on the one hand systematic differences, on the other, an excellent correlation between the test and comparison methods. From the spread of the data around the regression line in comparison with the 95% prediction intervals from the total RSD, sample-related effects and/or specificity problems were apparent and investigated.

LC-atmospheric pressure chemical ionization-MS/ MS analysis of multiple illicit drugs, methadone, and their metabolites in oral fluid following protein precipitation.

A quantitative LC-APCI-MS/MS method for simultaneous determination of multiple illicit drugs, methadone, and their metabolites in oral fluid was developed and validated. Sample pretreatment was limited to acetonitrile protein precipitation. LC separation was performed in 25.5 min, with a total analysis time of 35 min. Identification and quantitation were based on selected reaction monitoring. Calibration by linear regression analysis utilized deuterated internal standards and a weighing factor 1/x. Limits of detection and lower limits of quantitation (LOQ) were established between 0.25 and 5 ng/ mL and 0.5-10 ng/mL, respectively. linearity was obtained with an average correlation coefficient (R2) of >0.99, over a dynamic range from the LOQ up to maximum 500 ng/mL The method demonstrated good accuracy, intra- and interbatch precision, recovery, and stability for all compounds. No oral fluid matrix effect was observed throughout the chromatographic run. Protein precipitation provided a fast and simple sample pretreatment, while LC-APCI-MS/MS proved to be a sensitive and rugged quantitative method for multiple illicit and legal drugs in oral fluid. The method proved to be suitable for the evaluation of oral fluid as an alternative matrix to urine for monitoring illicit drug use and for determining oral fluid methadone concentrations in pregnant opiate and/or cocaine addicts.

Analysis of benzo[a]pyrene diol epoxide-DNA adducts by capillary zone electrophoresis- electrospray ionization-mass spectrometry in conjunction with sample stacking.

Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)-ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)(-) at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)(-) at m/z 632] and a BPDE-dCMP adduct [(M-H)(-) at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH(3) from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH(2) position of the nucleobase. So, the three adducts could be identified as BPDE-N(2)-dGMP, BPDE-N(6)-dAMP and BPDE-N(4)-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.

No compound a formation with Superia during minimal-flow sevoflurane anesthesia: a comparison with Sofnolime.

UNLABELLED: There is concern about the toxicity of Compound (Co) A. Absorbents differ in the production of Co A during minimal-flow sevoflurane anesthesia. Strong alkali-free Amsorb does not produce Co A. It was our aim to study Superia, another new NaOH- and KOH-free CO(2) absorbent, in minimal-flow anesthesia, compared with KOH-free Sofnolime. After Ethics Committee approval, 14 consenting adult patients were included randomly by using Superia or Sofnolime as the CO(2) absorbent in the compact 750-mL canister of an ADU ventilator. After propofol and remifentanil administration, sevoflurane was given in oxygen and air (500 mL/min; fraction of inspired oxygen, 0.4), aiming at an end-tidal concentration of 2.3%-2.5%; ventilation aimed for 33-35 mm Hg PETCO(2). Compound A inspired (Co A(insp)) and expired (Co A(exp)) samples were taken for analysis, and canister temperatures were measured for 150 min. Statistical analysis was performed with the Friedman test or the Mann-Whitney U-test where appropriate. Correction for multiple testing was used. In the Superia group, no significant amount of Co A was formed, whereas in the Sofnolime group, maximum median (range) inspiratory values of 25 ppm (16 ppm) were found. The intergroup difference was P < 0.05. No difference was noticed between the two groups for the canister CO(2) absorbent temperature. IMPLICATIONS: During minimal-flow 2.3%-2.5% end-tidal sevoflurane, no compound A (Co A) is formed with the NaOH- and KOH-free CO(2) absorbent Superia. Although Co A values with KOH-free Sofnolime are still within reported safe limits, Superia is definitely an alternative for safe clinical practice.

Simultaneous, quantitative determination of opiates, amphetamines, cocaine and benzoylecgonine in oral fluid by liquid chromatography quadrupole-time-of-flight mass spectrometry.

A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 micro of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66 x 10(-5)-1.48 x 10(-5) M) for all compounds. Internal standards were used to generate quadratic calibration curves (r(2)>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.

Reference measurement systems in clinical chemistry.

BACKGROUND: In clinical chemistry, traceability of measurements is of high priority. METHODS: In this literature review, current recommendations on the process of establishing traceability (or standardization) are critically discussed. RESULTS: Traceability is to be established to the highest international standards by a comprehensive reference measurement system. Elementary to this system are a metrological basis, a measurement unit system, i.e., the Système International d'Unités (SI), its embodiment by a material standard and a calibration hierarchy for transfer of accuracy/trueness to the manufacturer's product calibrators and routine methods. However, for analytes lacking an unequivocally recognized entity, the International Unit (IU) and International Standard (IS) concept have been developed. On this basis, the review distinguishes between traceability of SI- and IU-analytes. CONCLUSIONS: SI-traceability, exemplified by cortisol, is straightforward. However, special attention is needed for "free analytes" and "analyte families". For traceability of IU-analytes, exemplified by hCG, the standardization process passes different phases in function of the history of the analyte (discovery, ISs, measurement by bioassays/immunoassays, complete structure elucidation). However, perspectives to the development of an SI-based reference measurement system are realistic. Last but not least, for successful global implementation of the standardization process, consensus of all major players in the field will be required.

Distribution study of 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine in a fatal overdose.

In this study, regional tissue distributions of the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") and its metabolite 3,4-methylenedioxyamphetamine (MDA) in a fatal overdose are presented. Quantitation of MDMA and MDA levels occurred in blood samples taken centrally (right and left heart and main adjacent great vessels) and peripherally (subclavian and femoral blood). In addition, MDMA and MDA concentrations were determined in cardiac and iliopsoas muscle, both lungs, liver, both kidneys, spleen, the four brain lobes, cerebellum and brainstem, and adipose tissue. Finally, MDMA and MDA levels were determined in serum, vitreous humor, urine, and bile. For all samples, a fully validated high-pressure liquid chromatography procedure with fluorescence detection was used. The found substances were also identified with liquid chromatography-tandem mass spectrometry. Our data confirm that blood sampling from an isolated peripheral vein is recommended for MDMA and MDA. In addition, the vitreous humor MDMA level indicates that this fluid can be an interesting alternative when a suitable blood sample is missing. Considering the substantial differences in concentrations in blood samples taken from various sites in the body and the high levels in some tissues (e.g., in liver), we concluded that the influence of postmortem redistribution should be taken into account in the interpretation of toxicological data when an appropriate peripheral sample cannot be obtained or when blood samples are not available because of putrefaction.

Determination of paramethoxyamphetamine and other amphetamine-related designer drugs by liquid chromatography/sonic spray ionization mass spectrometry.

Paramethoxyamphetamine (PMA) is an amphetamine-like designer drug that has emerged recently on the European illicit drug market. This drug has a wicked reputation, as a number of lethal intoxications have occurred. A method using high-performance liquid chromatography coupled to ion trap based mass spectrometry (LC/MS) is described for the determination of this compound together with 3,4-methylenedioxymethamphetamine (XTC or MDMA), amphetamine and 3,4-methylenedioxyamphetamine (MDA) in human matrices. A liquid/liquid extraction (LLE) was applied to whole blood, urine and postmortem tissues. Reversed-phase liquid chromatography was performed on a narrow-bore phenyl-type column at a flow rate of 0.3 mL/min. A switch box allowed disposal of early-eluting irrelevant material to waste, protecting the mass spectrometer from contamination. The column effluent was directed into an ion trap mass spectrometer by a sonic spray ionization (SSI) interface. The method was validated for all three matrices, proving the applicability of SSI even when dealing with complex biological matrices. The within-and between-day precisions were less than 17.5% and accuracy was below 16.2%. Weighted (1/x) quadratic calibration curves were generated ranging from 10 to 1000 ng/mL (blood and urine) or 20 to 2000 ng/g (tissue) and correlation coefficients (r(2)) always exceeded 0.995. In addition, the mass spectrum of PMA is given together with a proposed fragmentation pattern for the obtained LC/MS spectrum. This information can be useful for future identification of PMA with LC/MS in biological matrices as well as in confiscated powders or tablets.

Quantitative determination of n-propane, iso-butane, and n-butane by headspace GC-MS in intoxications by inhalation of lighter fluid.

This report describes a fully elaborated and validated method for quantitation of the hydrocarbons n-propane, iso-butane, and n-butane in blood samples. The newly developed analytical procedure is suitable for both emergency cases and forensic medicine investigations. Its practical applicability is illustrated with a forensic blood sample after acute inhalative intoxication with lighter fluid; case history and toxicological findings are included. Identification and quantitation of the analytes were performed using static headspace extraction combined with gas chromatography-mass spectrometry. In order to reconcile the large gas volumes injected (0.5 mL) with the narrowbore capillary column and thus achieve preconcentration, cold trapping on a Tenax sorbent followed by flash desorption was applied. Adequate retention and separation were achieved isothermally at 35 degrees C on a thick-film capillary column. Sample preparation was kept to a strict minimum and involved simply adding 2.5 microL of a liquid solution of 1,1,2-trichlorotrifluoroethane in t-butyl-methylether as an internal standard to aliquots of blood in a capped vial. Standards were created by volumetric dilution departing from a gravimetrically prepared calibration gas mixture composed of 0.3% of n-propane, 0.7% of iso-butane, and 0.8% of n-butane in nitrogen. In the forensic blood sample, the following concentrations were measured: 90.0 microg/L for n-propane, 246 microg/L for iso-butane, and 846 microg/L for n-butane.