Methadone concentrations in blood, plasma, and oral fluid determined by isotope-dilution gas chromatography-mass spectrometry.

Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography-mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate-buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N = 5) were 1.27 and 1.21, respectively. In clinical samples from patients (N = 46), the concentrations of MTD in plasma and whole blood were highly correlated (r = 0.92, p < 0.001) and mean (median) plasma/blood distribution ratios were 1.43 (1.41). The correlations between MTD in OF and plasma (r = 0.46) and OF and blood (r = 0.52) were also statistically significant (p < 0.001) and the mean OF/plasma and OF/blood distribution ratios were 0.55 and 0.77, respectively. The MTD concentration in OF decreased as salivary pH increased (more basic). These results will prove useful in clinical and forensic medicine when MTD concentrations in alternative specimens are compared and contrasted.

Detector response and intensity cross-contribution as contributing factors to the observed non-linear calibration curves in mass spectrometric analysis.

It is a common knowledge that detector fatigue causes a calibration curve to deviate from the preferred linear relationship at the higher concentration end. With the adaptation of an isotopically labeled analog of the analyte as the internal standard (IS), cross-contribution (CC) of the intensities monitored for the ions designating the analyte and the IS can also result in a non-linear relationship at both ends. A novel approach developed to assess 'the extent and the effect of [CC]… in quantitative GC-MS analysis' can be extended (a) to examine whether a specific set of CC values is accurate; and (b) to differentiate whether the observed non-linear calibration curve is caused by detector fatigue or the CC phenomenon. Data derived from the exemplar secobarbital (SB)/SB-d(5) system (as di-butyl-derivatives) are used to illustrate this novel approach. Comparing the non-linear nature of calibration data that are empirically observed to that derived from theoretical calculation (with the incorporation of adjustment resulting from the ion CC phenomenon), supports the conclusions that (a) both CC and detector fatigue contribute significantly to the observed non-linear nature of the calibration curve based on ion-pair m/z 207/212; and (b) detector fatigue is the dominating contributor when the calibration curve is based on ion-pair m/z 263/268.

Methylglyoxal-mediated anxiolysis involves increased protein modification and elevated expression of glyoxalase 1 in the brain.

Methylglyoxal (MG) is a highly reactive metabolite that forms adducts with basic amino acid side chains in proteins. MG is degraded by glyoxalase1 (GLO1), an enzyme shown to be differentially expressed in several mouse models of anxiety-related behavior. As yet, molecular mechanisms by which altered GLO1 expression influences emotionality have not been elucidated. Here we report that both MG concentration and protein modification are altered in brain tissue of a mouse model for trait anxiety, with elevated levels in low anxiety-related behavior relative to high anxiety-related behavior animals. Accordingly, repeated intracerebroventricular injections of MG mediated anxiolysis in inbred high anxiety-related behavior and outbred CD1 mice. We found that anxiolytic-like properties of MG were independent of GLO1 expression. In contrast, antidepressant-like properties of intracerebroventricular MG were suppressed in CD1 mice carrying extra copies of the GLO1 gene. Moreover, MG treatment increased expression of GLO1 only in CD1 mice that did not have extra copies of GLO1. Taken together, these results suggest that the MG levels in brain are negatively correlated with anxiety. Thereby, we identified a novel molecular mechanism for anxiety-related behavior in mice that may help to elucidate genesis of psychiatric disorders in humans.

A novel derivatization approach for simultaneous determination of glyoxal, methylglyoxal, and 3-deoxyglucosone in plasma by gas chromatography-mass spectrometry.

A two-step derivatization approach has been developed to enable the simultaneous analysis of glyoxal, methylglyoxal, and 3-deoxyglucosone by the most efficient and widely applied GC-MS methodology. These three analytes are reactive carbonyl compounds associated with the formation of advanced glycation and lipoxidation end products, a process thought to contribute to uremic toxicity and referred to as "carbonyl stress". Effective analysis of these compounds would facilitate understanding these compounds' role in diabetes-related complications. Plasma samples were deproteinized by acetonitrile, followed by a two-step derivatization approach. Pooled plasma samples from healthy individuals were used as the "blank" for preparing calibration standards. The concentrations of the analytes in the "blank" were first determined by standard addition method. Calibration parameters were accordingly established and used to analyze these compounds in plasma samples collected from healthy individuals and diabetic patients. Analytical findings are comparable with those reported in the literature. Quantitation data can be further improved by making available and using isotopically labeled analogs of these analytes as the internal standards.

A novel approach to evaluate the extent and the effect of cross-contribution to the intensity of ions designating the analyte and the internal standard in quantitative GC-MS analysis.

In gas chromatography-mass spectrometry methods of analysis adopting the analyte's isotopic analog as the internal standard (IS), the cross-contribution (CC) phenomenon -- contribution of IS to the intensities of the ions designating the analyte, and vice versa -- has been demonstrated to affect the quantitation data. A novel approach based on the deviations of the empirically observed concentrations of a set of standards was developed to assess the accuracy of the empirically derived CC data. This approach demonstrated that normalization of ion intensities derived from the analyte and the IS generates reliable CC data. It further demonstrated that an ion-pair (designating the analyte and the IS) with approximately 5% or higher CC will result in a very limited linear calibration range.

Evaluation of various derivatization approaches for gas chromatography-mass spectrometry analysis of buprenorphine and norbuprenorphine.

Various chemical derivatization approaches have been adapted for the analysis of buprenorphine and its major metabolite (norbuprenorphine) by GC-MS based methodologies. These approaches included alkylation, acylation, and silylation resulting in the formation of methyl, acetyl, trifluoroacetyl, pentafluoropropionyl, heptafluorobutyryl, and trimethylsilyl derivatives. This study conducted a comprehensive evaluation on the merits of these approaches based on the following criteria: reaction yields and ionization efficiency of the derivatization products; chromatographic characteristics; and cross-contributions to the intensities of ions designating the analytes and the internal standards. Under acidic derivatization conditions, the analytes could form three artifact products. Overall, derivatization by acetyl anhydride resulted in best performance characteristics.

GC-MS analysis of multiply derivatized opioids in urine.

Opiates such as hydrocodone, hydromorphone, oxycodone, noroxycodone, and oxymorphone reportedly may interfere with the analysis of morphine and codeine. The analysis of these compounds themselves also is an important issue. Thus, double derivatization approaches utilizing methoxyamine and hydroxylamine to first form oxime products with keto-opiates, followed by the derivatization with trimethylsilyl (TMS) or propionyl groups, have been developed for the simultaneous analysis of these compounds. However, these studies have not included all compounds of interest and resulted in inadequate chromatographic resolution or significant intensity cross-contribution between the ions designating the analyte and its deuterated internal standard for certain compounds. By exploring three-step derivatization approaches with the combination of various derivatization groups and orders, this study concluded that application of methoxyimino/propionyl/TMS groups, in the order listed, facilitated the simultaneous analysis of eight opiates (morphine, 6-acetylmorphine, hydromorphone, oxymorphone, codeine, hydrocodone, oxycodone and noroxycodone) in urine samples, achieving satisfactory limits of quantitation and detection. In addition, the adapted approach resulted in two usable products for morphine and codeine providing alternatives, should interferences render any of these products non-usable.