Amrik S. Walia

Simultaneous analysis of amphetamine, methamphetamine, and 3, 4-methylenedioxymethamphetamine (MDMA) in urine samples by solid-phase extraction, derivatization, and gas chromatography/mass spectrometry

A rapid and effective solid-phase extraction procedure using Bond Elute Certify bonded silica sorbent cartridges was adopted to extract amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) from urine samples. The extract was derivatized with trichloroacetic anhydride prior to gas chromatography/mass spectrometry (GC/MS) analysis with selected ion monitoring of the following ions: 190, 91, 188; 204, 91, 202; 162, 135, 202; 194, 123; and 211, 209 for the derivatized amphetamine, methamphetamine, MDMA, d5-amphetamine, and d9-methamphetamine, respectively. The first of the ions listed for each compound was used for quantitation. The compound d5-amphetamine was used as the internal standard for amphetamine, and d9-methamphetamine was used for methamphetamine and MDMA. Results showed a higher than 65% recovery and a reproducibility with less than a 5% coefficient of variation. When a sample size of 2 mL was used, the lowest detectable concentration was about 50 ng/mL, and a near-perfect fit can be obtained (within the 250 to 4000-ng/mL concentration range studied) using a second-order polynomial model.

Effects of retinoids on macrophage function and IL-1 activity.

The effects of three retinoids, all‐trans‐retinoic acid (RA), 13‐cis‐retinoic acid (cRA), and N‐(4‐hydroxyphenyl) ratinamide (4‐HPR), on macrophage function were evaluated. In vitro, RA, cRA, and 4‐HPR caused a greater than twofold increase in phagocytosis of IgG‐sensitized bovine erthrocytes (IgG‐ORBC) by a mouse macrophage cell line (RAW). Significant increases in phagocytosis were produced by retinoid concentrations as low as 2 x 10‐10 M. RA also significantly increased phagocytosis of IgG‐sensitized ORBC by BALB/c peritoneal macrophages In vitro. The ability of RAW macrophages to bind IgG‐ORBC was significantly increased by 10‐6 to 10‐14 M RA. The potentiation of myogenic responses of spleen cells to Con A and PWM by RA was relatively independent of macrophage function, i.e., splenocytes that were macrophage‐depleted were responsive to the potentiating effects of RA. The effects of retinoids on T‐cell‐dependent B‐cell mitogenesis induced by PWM appeared not to be dependent on their previously reported capacity to alter prostaglandin synthesis. Treatment of spleen cells with 10‐6 M indomethacin did not abolish the potentiating effects of RA. However, RA in a dose‐dependent fashion increased IL‐1 activity at the level of the target T‐cell. The greatest potentiation of IL‐1 activity was at 10‐8 M RA. These results show that retinoids can modulate macrophage function at two different levels: potentiation of phagocytosis and potentiation of IL‐1 activity at the level of the T‐cell.

Improved Gas Chromatography/Mass Spectrometry Analysis of Barbiturates in Urine Using Centrifuge-Based Solid-Phase Extraction, Methylation, with d5-Pentobarbital as Internal Standard

Effective solid-phase extraction, derivatization, and GC/MS procedures are developed for the simultaneous determinations of butalbital, amobarbital, pentobarbital, and secobarbital, using a deuterated pentobarbital (d5-pentobarbital) as the internal standard. Buffered (pH 7) urine samples were extracted with Bond Elute Certify II cartridge. Iodomethane/tetramethylammonium hydroxide in dimethylsulfoxide was used for methylation, while a HP 5970 MSD equipped with a 13 m J & W DB-5 column (5% phenyl polysiloxane phase) and the Thru-Put Target software package were used for GC/MS analysis and data processing. This protocol was found to be superior, in both chromatographic performance characteristics and quantitation results, over a liquid-liquid extraction procedure without derivatization using hexobarbital as the internal standard. Extraction recoveries observed from control samples containing four barbiturates range from 80% to 90%. Good one-point calibration data are obtained for all four barbiturates in the 50 to 3200 ng/mL range. Interestingly, the one-point calibration data for pentobarbital are inferior to the other three barbiturates--due to interference from the internal standard (d5-pentobarbital). The calibration data of pentobarbital are best described by a hyperbolic curve regression model. Precision data (% CV) for GC/MS analysis, over-all procedure, and day-to-day performance are approximately 2.0%, 6.0%, and 8.0%, respectively. With the use of a 2 mL sample size, the attainable detection limit is approximately 20 ng/mL.

Functional aspects of IgM and IgG Fc receptors on murine T lymphocytes.

All mammalian cells receive regulating signals via surface receptors of various kinds. These receptors specifically recognize molecules like hormones, transmittor substances, pharmacological reagents, mitogens and viruses. Lymphocytes have receptors for antigens (sig), activated complement components (C3receptors), various regulatory molecules released from cells activated by mitogens or viruses (e.g. interferon and TCGF), and finally receptors for the Fc part of immunoglobuiin molecules (FcR). FcR exist on a variety of different cell types where they may or may not be involved in the specific function of the cell. Among the most striking examples of FcR mediated cellular functions are Type I hypersensitivity mediated by mast cells via their FcR for IgF, transplacental transport of IgG, antibody-dependent cell mediated cytotoxicity (ADCC) by killer (K) cells, phagocytosis of antibody coated particles and modulation of immune responses (positive or negative) by immune complexes. The present review deals with studies on murine T lymphocytes bearing surface FcR. We will not limit the term receptor only to structures which mediate a functional change of the cells, but will rather use the term receptor

In vitro effects of retinoids on murine thymus-dependent and thymus-lndependent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.

Correlations on radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay of cannabis metabolites with gas chromatography/mass spectrometry analysis of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine specimens

Results obtained from three commercial immunoassay kits, Abuscreen, TDx, and EMIT, commonly used for the initial test of urine cannabinoids (and metabolites) were correlated with the 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (9-THC-COOH) concentration as determined by GC/MS. Correlation coefficients obtained based on 26 (out of 1359 total sample population) highly relevant samples, are 0.601 and 0.438 for Abuscreen and TDx. Correlation coefficients obtained from a parallel study on a different set of 47 (out of 5070 total sample population) highly relevant specimens are 0.658 and 0.575 for Abuscreen and Emit. The immunoassay concentration levels, that correspond to the commonly used 15 ng/ml GC/MS cutoff value for 9-THC-COOH, as calculated from the regression equations are 82 ng/ml and 75 ng/ml for TDx and EMIT and 120 ng/ml and 72 ng/ml for Abuscreen manufactured at two different time periods. The difference of these calculated corresponding concentrations provides quantitative evidence of the reagent specificity differences.

In vitro effect of ethanol on cell-mediated cytotoxicity by murine spleen cells

The effects of ethanol on murine spleen cell-mediated lysis have been studied. Concentrations of 5.5-176 mM ethanol produced progressive inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC). Binding of spleen cells to antibody-sensitized target cells was not inhibited by comparable concentrations of ethanol. Kinetic analysis revealed decreased rates of lysis with increasing concentrations of ethanol. Changes of effector to target cell ratios revealed an inhibition of maximum lysis and decreased lytic efficiency in the presence of 88 mM ethanol. Preincubation experiments showed the inhibitory effect of ethanol to be reversible. Macrophage-depleted spleen cells appeared to be as susceptible to inhibition by ethanol as unfractionated spleen cells. Ethanol also inhibited natural killer and alloimmune cytotoxic T cell activity. The ADCC data were analysed by using a mathematical model which incorporates the kinetics of lysis, dose-response relationships, heterogeneity of the lytic effectors, reversibility of inhibition and ethanol loss during incubation. An inhibition constant (KI) of 373 mM-2 when two ethanol molecules interact with the site of inhibition was calculated. 50% inhibition of lysis is produced by 52 mM (0.24%) ethanol. The results are consistent with a model which assumes that lysis is due to a critical number of interactions which ultimately trigger the lytic event. Alcohol interferes with lysis by reacting with sites which are required for triggering the lytic event. Although the molecular details of the mechanism of inhibition are as yet undefined, we infer that ethanol inhibits ADCC at the programming for lysis or the lethal hit stages.

Isotopic Analogue as the Internal Standard for Quantitative Determination of Benzoylecgonine: Concerns with Isotopic Purity and Concentration Level

Empirical data is used to demonstrate the observation and quantification of benzoylecgonine in negative test samples when high concentrations of deuterated benzoylecgonine are used as the internal standard in the assay process. On the quantitative determination of true positive samples, inaccuracy introduced by the isotopic impurity of the internal standard is calculated as a function of the impurity and the concentration levels of the internal standard used.

Simultaneous Gas Chromatography/Mass Spectrometry Assay of Methadone and 2-Ethyl-1, 5-Dimethyl-3,3-Diphenylpyrrolidine (EDDP) in Urine

An efficient extraction and gas chromatography/mass spectrometry (GC/MS) procedure has been developed for the simultaneous determination of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine in urine samples. The merits of this procedure include (1) effective high-volume sample processing; (2) excellent gas chromatography characteristics; (3) high precision for quantitative methadone determination--1.0% coefficient of variation (CV) for GC/MS injection replicates and 1.2% for extraction replicates; (4) excellent linearity within the range (0 to 1200 ng/mL) studied; and (5) adequate detection limits (50 ng/mL) for most practical purposes. The detection limit for methadone may be improved 40-fold by using a different internal standard.

Antibody-dependent cell-mediated cytotoxicity in the Moloney sarcoma virus system: No requirement for exogenous C5☆

Abstract It has been demonstrated that IgM antibody-dependent cell-mediated cytotoxicity (ADCC) does not require exogenous C5 in the Moloney sarcoma virus (M-MuSV) system by employing A J (C5 deficient) mice as a source of antibody, lymphocytes, and serum for support of cultures. The A J mice injected with the M-MuSV developed tumors within 5 days which were regressed by 16–20 days. The pooled antisera from these regressor animals were fractionated on Sephadex G-200 to obtain the 19 and 7 S fractions. The capability of the fractions to induce ADCC by spleen, lymph node, and thymus cells was tested against cells possessing the relevant virus-induced antigens and control cells lacking these antigens. The assays were carried out in medium supplemented with heat inactivated A J serum. The results indicate that C5-deficient A J mice are capable of regressing M-MuSV-induced tumors and, therefore, that exogenous C5 is not required for this in vivo response. These mice produce both IgM and IgG with very high titers in ADCC against the specific target cells. Lymphocytes from the spleen, lymph node, and thymus of A J mice are active effector cells against IgM-sensitized target cells with titers being generally higher with thymocytes. IgG-sensitized target cells were lysed only by splenocytes and lymph node cells. The involvement of membrane-bound complement components was not determined in this study. The assay was conducted in heat-inactivated serum in the absence of exogenous C5, and we conclude that IgG- and IgM-ADCC do not require the classical or alternative pathway of complement fixation.