Determination of paclitaxel and metabolites in mouse plasma,tissues, urine and faeces by semi-automated reversed-phase high-performance liquid chromatography

We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6α,3′-p-dihydroxypaclitaxel, 3′-p-hydroxypaclitaxel and 6α-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1–0.2 g/ml) in bovine serum albumin (40 g/I) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200–1000 μl of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut column. 2′-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 ot 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 μl of sample. In tissues, faeces and urine the LLQs are 25–100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 μl (faeces: 200 μl) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25–100 000 ng/ml for paclitaxel and 25–500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.     

Multiplex single molecule counting technology used to generate interleukin 4, interleukin 6, and interleukin 10 reference limits

Detecting biomarkers at pg/ml concentrations or below is, in many situations, critical for quantifying levels in healthy individuals as well as the changes that can occur in the progression of disease states. The ability to detect multiple biomarkers from the same sample allows for better diagnoses, more efficient testing, and lower volumes of sample required. Based on single molecule counting technology, a multiplex instrument was designed and built that is capable of detecting cytokines and other low-abundance proteins at sub-pg/ml quantities in human plasma samples. The multiplex single molecule counting instrument was used to generate 95% reference limits for interleukin 4 (IL-4, <0.61 pg/ml), interleukin 6 (IL-6, <6.53 pg/ml), and interleukin 10 (IL-10, <1.08 pg/ml) from 100 healthy human donor plasma samples, with more than 90% of IL-4 concentrations and 100% of IL-6 and IL-10 concentrations above the limit of detection.     

Attention deficit hyperactivity disorder: fine mapping supports linkage to 5p13, 6q12, 16p13, and 17p11

We completed fine mapping of nine positional candidate regions for attention-deficit/hyperactivity disorder (ADHD) in an extended population sample of 308 affected sibling pairs (ASPs), constituting the largest linkage sample of families with ADHD published to date. The candidate chromosomal regions were selected from all three published genomewide scans for ADHD, and fine mapping was done to comprehensively validate these positional candidate regions in our sample. Multipoint maximum LOD score (MLS) analysis yielded significant evidence of linkage on 6q12 (MLS 3.30; empiric P=.024) and 17p11 (MLS 3.63; empiric P=.015), as well as suggestive evidence on 5p13 (MLS 2.55; empiric P=.091). In conjunction with the previously reported significant linkage on the basis of fine mapping 16p13 in the same sample as this report, the analyses presented here indicate that four chromosomal regions--5p13, 6q12, 16p13, and 17p11--are likely to harbor susceptibility genes for ADHD. The refinement of linkage within each of these regions lays the foundation for subsequent investigations using association methods to detect risk genes of moderate effect size.     

Molecular analysis to FUT-1, 2, 3 and ABO genotyping may instead of serological typing to diagnose para-Bombay in Chinese

The aim of this study was to evaluate the consistency between serotyping and molecular analysis in Chinese with para-Bombay. The molecular analysis of gene fragments in FUT-1, FUT-2, FUT-3 and ABO genotyping and serotyping were used including a saliva test to examine the A, B, H substance and an absorption elution test to examine the A, B, H; and further routine tests including ABO, H and Lewis phenotype. From eleven samples with anti-H negative, 10 samples were confirmed with para-Bombay by sequencing to FUT-1, from which six samples were 547-548delAG, three samples were 880TT deletion, one sample was 35C>T and one sample was 649G>T heterozygous (h7, China) as carrier. The sequencing to FUT-2 confirmed 357C>T in 11 samples, meaning H, A and B substance was secreted in saliva except for one sample which occurred 385A>T (I129F) heterozygous, which is a weak secretor. The FUT-3 sequence result demonstrated four samples with heterozygous mutations to 59T>G (L20R) combined with 508G>A (G170S) and seven samples without mutations in FUT-3 gene fragment same as reference. The consistency between sequencing with FUT-1/FUT-2 and serotyping by anti-H reported an identical result, except for one sample, which interestingly showed the H/h7 carrier with serotyping negative to anti-H. The result of sequencing with FUT-2/FUT-3 and Lewis phenotyping also reported a complete consistency. The saliva test to A, B, H substance and absorption elution test examining the A, B, H antigens on the surface of red blood cells completely matched the ABO exon 6, 7 sequence results. The sequencing of FUT-1, FUT-2, FUT-3 and ABO exon 6, 7 may become a useful tool to confirm the para-Bombay blood type.     

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

A comparison of chloride, bromide, and sucrose dilution volumes in neonatal pigs

Use of 36Cl, 82Br, and [3H]sucrose to estimate extracellular water volume was evaluated in 14 piglets (7-14 days old). 36Cl and 82Br were distributed in approximately the same volume, but a period of 5-6 hr after injection was required to reach equilibrium in the neonatal pig. Dilution volumes calculated before equilibration (2-5 hr) for 36Cl (326 +/- 11 ml/kg) and 82Br (328 +/- 13 ml/kg) were different from equilibration (6-8 hr) phase volumes (356 +/- 13 ml/kg and 355 +/- 13 ml/kg, respectively; P less than 0.001). A 3-hr sample estimated the same volume distribution calculated by extrapolation of the 6- to 8-hr period because of the relationship between the two slopes of the plasma clearance curves. After the 82Br and 36Cl had achieved equilibration, each was distributed in a volume equivalent to total body chloride space (362 +/- 29 ml/kg) measured by neutron activation; no statistical differences were found (P = 0.6). The early equilibration phase measured a 10% smaller, faster exchangeable fraction of total body Cl. Sucrose dilution volume (332 +/- 19 ml/kg) required multiple plasma samples for extrapolation and measured a dilution volume 7% smaller (P less than 0.05) than total body chloride space.     

Rapid evaluation of ant diversity in land use systems in southern Bahia, Brazil

We aimed to compare the soil ant diversity in different land use systems from Atlantic Forest area, in Southern Bahia state, Brazil. The ants were sampled in 16 sites: two primary forest sites (un-logged forest); three young secondary forests (<8 years old); three intermediate secondary forests (8-20 years old); three old secondary forests (>20 years old); three Eucalyptus grandis plantations (3-7 years old), and two introduced pastures. Each site was sampled in three sampling points 15 m apart, and distant over 50 m from the site edge. In each sampling point we gathered the litter from a 1 m2 and extracted the ants with Winkler extractors during 48h. We found 103 ant species from 29 genera and eight subfamilies. The five richest genera were Pheidole (19 species), Solenopsis (8), Apterostigma (10), Hypoponera (7) e Paratrechina (5). The highest ant richness density was found in the primary forest (7.4 species/sample; S = 37; n = 5); followed by the old secondary forest (5.33 species/sample; S = 48; n = 9); young secondary forest (5.25 species/sample; S = 42, n = 8); eucalyptus plantation (4.22 species/sample; S = 38, n = 9), intermediate secondary forest (3.5 species/sample; S = 35, n = 10, and introduced pasture (2.67 species/sample; S = 16, n = 6). The ecosystems with higher structural complexity showed the highest ant richness density by sample. Therefore, in the Atlantic Forest region, the eucalyptus plantation is a better alternative of land use to conserve the ant biodiversity than pastures, and quite similar to native secondary forests in ant community characteristics.     

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.     

Determination of the HIV integrase inhibitor, MK-0518 (raltegravir), in human plasma using 96-well liquid-liquid extraction and HPLC-MS/MS

An HPLC-MS/MS method was developed for the determination of MK-0518 (raltegravir), an HIV integrase inhibitor, in human plasma over the concentration range of 2-1000 ng/mL. Stable isotope labeled (13)C(6)-MK-0518 was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction with hexane:methylene chloride in the 96-well format with a 200 microL plasma sample size. The compounds were chromatographed on an Ace C(18) (50 x 3.0 mm, 3 microm, titanium frits) column with 42.5/57.5 (v/v %) 0.1mM EDTA in 0.1% formic acid/methanol mobile phase at a flow rate of 0.5 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for MK-0518 (m/z 445-->109) and (13)C(6)-MK-0518 (m/z 451-->367) on an Applied Biosystem API 4000 HPLC-MS/MS was used for quantitation. Intraday precision of standard curve concentrations in five different lots of control plasma was within 3.2%, while accuracy ranged from 94.8 to 106.8%. The mean extraction recovery of spiked plasma samples was 87%. Quality control (QC) samples were stored at -20 degrees C. Initial within day analysis showed QC accuracy within 7.5% of nominal with precision of 3.1% or less. The plasma QC samples were demonstrated to be stable for up to 23 months at -20 degrees C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development.     

Synergized subcritical-ultrasound-assisted aqueous two-phase extraction,purification, and characterization of Lentinus edodes polysaccharides

The effect of a synergized two-step subcritical water extraction (SWE) and simultaneous multi-frequency ultrasound-assisted alcohol/salt aqueous two-phase extraction (MFu-AATPE) on the yield, physicochemical, structural characteristics, and antioxidant activities of Lentinus edodes polysaccharides (LEPs) was investigated. Crude LEP extracted under subcritical extraction (31.14%) was purified using dual and triple frequency ultrasound-assisted aqueous two-phase systems giving a yield of 94.57% and 97% respectively. Compared with the dual-frequency sonication (20/40 kHz), the triple frequency sonication (20/40/60 kHz) gave maximum yield and improved the desalination rate by 33.6%. Congo red analysis revealed triple helix structures. Gas Chromatography analysis showed the existence of mannose, glucose, arabinose, xylose, and galactose whilst spectral similarities were observed through Fourier Transform Infrared Spectroscopy. The average molecular weight (M_w) was 83.6170 Da, 2384 Da and 2387 Da for control, dual and triple frequency samples respectively. Ultrasound treated polysaccharides displayed stronger antioxidant activities against the 2.2-diphenyl;-1 picrylhydrazyl (DPPH), 2, 2ʹ-azino-bis 3-ethylbenzothiazoline-6 sulfonic acid (ABTS) and hydroxyl radicals, thus demonstrating their potency in reducing oxidation. However, the microstructure of the triple frequency treated sample was altered compared to the dual-frequency sample. Therefore, this study demonstrated the efficacy of SWE in crude LEPs extraction and illustrated MFu-AATPE as an efficient technique that selectively isolates polysaccharides and enhances extraction efficiencies.     

An Examination of the Association between 5-HTTLPR,Combat Exposure,and PTSD Diagnosis among U.S. Veterans

Objective

To examine the association between the 5-HTTLPR polymorphism of the serotonin transporter (SLC6A4) gene, combat exposure, and posttraumatic stress disorder (PTSD) diagnosis and among two samples of combat-exposed veterans.

Method

The first sample included 550 non-Hispanic Black (NHB) combat-exposed veterans. The second sample included 555 non-Hispanic White (NHW) combat-exposed veterans. Participants were genotyped for the 5-HTTLPR/rs25531 variants of the SLC6A4 gene. A structured clinical interview was used to diagnose PTSD. Combat and civilian trauma exposure were assessed with validated self-report instruments. Logistic regression was used to test for main effects of 5-HTTLPR on PTSD diagnosis as well as gene x environment (GxE) interactions after adjusting for sex, ancestry proportion scores, civilian trauma exposure, and combat exposure.

Results

Within the NHB sample, a significant additive effect was observed for 5-HTTLPR (OR = 1.502, p = .0025), such that the odds of having a current diagnosis of PTSD increased by 1.502 for each additional S’ allele. No evidence for an association between 5-HTTLPR and PTSD was observed in the NHW sample. In addition, no evidence for combat x 5-HTTLPR effects were observed in either sample.

Conclusion

The present study suggests that there may be an association between 5-HTTLPR genotype and PTSD diagnosis among NHB veterans; however, no evidence for the hypothesized 5-HTTLPR x combat interaction was found.     

Analysis and quantification of ammonia-oxidizing bacteria community with amoA gene in sewage treatment plants

The analysis and quantification of ammonia-oxidizing bacteria (AOB) is crucial, as they initiate the biological removal of ammonia-nitrogen from sewage. Previous methods for analyzing the microbial community structure, which involve the plating of samples or culture media over agar plates, have been inadequate because many microorganisms found in a sewage plant are unculturable. In this study, to exclusively detect AOB, the analysis was carried out via denaturing gradient gel electrophoresis using a primer specific to the amoA gene, which is one of the functional genes known as ammonia monooxygenase. An AOB consortium (S1 sample) that could oxidize an unprecedented 100% of ammonia in 24 h was obtained from sewage sludge. In addition, real-time PCR was used to quantify the AOB. Results of the microbial community analysis in terms of carbon utilization ability of samples showed that the aeration tank water sample (S2), influent water sample (S3), and effluent water sample (S4) used all the 31 substrates considered, whereas the AOB consortium (S1) used only Tween 80, D-galacturonic acid, itaconic acid, D-malic acid, and L-serine after 192 h. The largest concentration of AOB was detected in S1 (7.6 × 10(6) copies/microliter), followed by S2 (3.2 × 10(6) copies/microliter), S4 (2.8 × 10(6) copies/microliter), and S3 (2.4 × 10(6) copies/microliter).     

Direct injection isocratic high-performance liquid chromatographic analysis of mitomycin C in plasma

A direct injection high-performance liquid chromatography method is described for the determination of mitomycin C (MMC) in human plasma. The stationary phase consisted of hydrophilic and hydrophobic functional groups covalently bound to silicone-coated silica beads (CAPCELL PAK MF Ph-1, 150×4.6 mm I.D., 5 μm). A mobile phase using 100% water gave a better separation of MMC from endogenous interferences as compared to a mobile phase with 12.5% acetonitrile and 2.5 mM phosphate buffer (pH 6.9). Using water as the eluent (1 ml/min) and UV detection at 365 nm, MMC was found to elute at 5.0 min with a peak width of 0.3 min, whereas endogenous interferences eluted before 3 min. Total assay time per sample was 6 min. Internal standard was not required because the recovery of MMC was nearly complete, about 90% from 20 to 5000 ng/ml. The standard curve was linear from 20 to 5000 ng/ml in plasma, and the intra- and inter-day variation was between 3 to 6%. The lower detection limit was 5 ng/ml with a 25 μl sample, which represent a two- to four-fold improvement over the 10 ng/ml detection limit by previous methods using liquid-liquid extraction and comparable sample size. The simplicity of this method, i.e., no sample extraction, no internal standard, 100% aqueous mobile phase, isocratic elution and short analysis time (6 min/sample), makes it suitable for large scale routine sample analysis, whereas its small sample volume requirement and high sensitivity are useful for pharmacokinetic studies in small animals where limited sample is available.     

Irish kefir-like grains: their structure, microbial composition and fermentation kinetics

The structure of six Irish kefir samples was studied in the electron microscope, and the microbial composition and fermentation kinetics during growth in 10% reconstituted skim milk at 21°C. The microbial composition of the six samples was similar; at the end of the fermentation the counts of lactococci, leuconostocs, lactobacilli, acetic acid bacteria and yeasts were 10⁹, 10⁸, 5 × 10⁶, 10⁵ and 10⁶ ml⁻¹ respectively; the levels of acetic acid bacteria and lactobacilli showed some intersample differences. Lactate was the major metabolite followed in order by ethanol, acetate and acetoin. The final concentrations of L-lactate produced (66–90 mmol kg⁻¹) were 10-fold higher than those of D-lactate. Acetate and ethanol concentrations varied from 4 to 14 and 2 to 40 mmol kg^−'1 respectively. The rates of citrate utilization and concentration of acetoin produced during growth differed between samples. Scanning electron microscopy showed not only variation between the interior and exterior of the sample but also large variation between different sections of the interiors and exteriors of the same sample. Long and short, and straight and curved rods and yeasts were seen in all samples, the curved rods observed in the interior, but lactococci were seen on the surface of only one sample. There were no gross differences in structure between samples.     

The use of liposomes for detection of the surface lipopolysaccharide antigen, Vibrio cholerae cells, and antibodies against them

A test system for determination of Vibrio cholerae cells, surface O-antigen, and antibodies against them was developed on the basis of complement-dependent lysis of liposomes sensitized by the lipopolysaccharide-dependent antigen from Vibrio cholerae 569B. The factors that affect the function of the liposomal reagent were studied, and the conditions for detecting antibodies and antigenic material were optimized. This system is highly specific and sensitive to be used for the determination of anticholeraic antibodies (30-50 times as effective as agglutination tests), lipopolysaccharide antigen (100 ng/ml, which corresponded to 3.0 ng of lipopolysaccharide in the sample studied), and Vibrio cholerae cells (3.3 x 10(7) m.b./ml, which corresponded to 10(6) m.b. in sample). It takes 30-40 min to detect the lipopolysaccharide antigen and 90 min to detect V. cholerae cells.     

Solid phase extraction--non-aqueous capillary electrophoresis for determination of metformin, phenformin and glyburide in human plasma

Solid phase extraction (SPE) was coupled at line to capillary electrophoresis (CE) for the determination of three basic and neutral diabetic drugs (metformin, phenformin and glyburide) in human plasma. The SPE procedure employed a C(18) cartridge to remove most of the water and proteins from the plasma sample. Analyte detectability was increased due to trace enrichment during the SPE process. Elution of metformin, phenformin and glyburide was achieved with methanol+3% acetic acid. CE analysis was performed using a non-aqueous buffer, acetonitrile+5mM ammonium acetate+5% acetic acid, which afforded rapid separation of metformin from phenformin within 3 min. Glyburide, with a migration time longer than 6 min, did not cause any interference. The present SPE-CE method, with an electrokinetic injection time of 6s and UV detection at 240 nm, was useful for monitoring down to 1 microg/mL of metformin and phenformin in human plasma. When the electrokinetic injection time was increased to 36s, the detection limits were improved to 12 ng/mL for metformin and 6 ng/mL for phenformin.     

A genetic association study of chromosome 11q22-24 in two different samples implicates the FXYD6 gene, encoding phosphohippolin, in susceptibility to schizophrenia

Previous linkage analyses of families with multiple cases of schizophrenia by us and others have confirmed the involvement of the chromosome 11q22-24 region in the etiology of schizophrenia, with LOD scores of 3.4 and 3.1. We now report fine mapping of a susceptibility gene in the 11q22-24 region, determined on the basis of a University College London (UCL) sample of 496 cases and 488 supernormal controls. Confirmation was then performed by the study of an Aberdeen sample consisting of 858 cases and 591 controls (for a total of 2,433 individuals: 1,354 with schizophrenia and 1,079 controls). Seven microsatellite or single-nucleotide polymorphism (SNP) markers localized within or near the FXYD6 gene showed empirically significant allelic associations with schizophrenia in the UCL sample (for D11S1998, P=.021; for rs3168238, P=.009; for TTTC20.2, P=.048; for rs1815774, P=.049; for rs4938445, P=.010; for rs4938446, P=.025; for rs497768, P=.023). Several haplotypes were also found to be associated with schizophrenia; for example, haplotype Hap-F21 comprising markers rs10790212-rs4938445-rs497768 was found to be associated with schizophrenia, by a global permutation test (P=.002). Positive markers in the UCL sample were then genotyped in the Aberdeen sample. Two of these SNPs were found to be associated with schizophrenia in the Scottish sample (for rs4938445, P=.044; for rs497768, P=.037). The Hap-F21 haplotype also showed significant association with schizophrenia in the Aberdeen sample, with the same alleles being associated (P=.013). The FXYD6 gene encodes a protein called "phosphohippolin" that is highly expressed in regions of the brain thought to be involved in schizophrenia. The protein functions by modulating the kinetic properties of Na,K-ATPase to the specific physiological requirements of the tissue. Etiological base-pair changes in FXYD6 or in associated promoter/control regions are likely to cause abnormal function or expression of phosphohippolin and to increase genetic susceptibility to schizophrenia.     

Reversed-phase liquid chromatography method to determine COL-3, a matrix metalloproteinase inhibitor, in biological samples

A reversed-phase high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline (COL-3), a matrix metalloproteinase (MMPs) inhibitor, in rat serum. This simple, sensitive, rapid and reproducible assay involved a preliminary serum deproteinization by adding a mixture of acetonitrile-methanol-0.5 M oxalic acid (70:20:10 (v/v)), as the combined precipitant and metal blocking agent, into serum samples (2:1 (v/v)). An aliquot (20 microl) of the supernatant was injected into the HPLC system linked to a Waters XTerra RP(18) column (150 mm x 4.6 mm i.d., particle size 5 microm). The compound was eluted by a mixture of acetonitrile-methanol-0.01 M oxalic acid (40:10:50 (v/v), pH 2.00), as the mobile phase, and detected at the wavelength of 350 nm. The total running time was 10 min. The low and high concentration calibration curves were linear in the range of 50-1200 ng/ml and 1200-12,000 ng/ml, respectively. The intra- and inter-day coefficients of variation at three quality control concentrations of 100, 1200, and 12,000 ng/ml were all less than 6%, while the percent error ranged from -2.5 to 6.6%. The limit of quantitation (LOQ) for COL-3 in serum was 50 ng/ml. This assay was successfully employed to study the serum concentration-time profiles of COL-3 after its intravenous and oral administration in rats. The method with some minor modifications in sample pretreatment was also applicable to the determination of the concentrations of COL-3 in rat bile, urine and feces.     

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.     

Sequential GC/MS analysis of sialic acids,monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives

A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.