Analysis of ginkgolides and bilobalide in food products using LC-APCI-MS

A method was developed for the extraction and quantification of five marker compounds characteristic of Ginkgo biloba. Five ginkgo terpene trilactones: bilobalide and ginkgolides A, B, C, and J, were selected as marker compounds for this study. Initial studies produced a simple methanol extraction method for determination of gingko markers in solid dietary supplements. Five dietary supplements were analyzed and the results were later compared to the concentrations detected in the analysis of beverages. Beverage samples were prepared by extracting the ginkgo terpene trilactones using an optimized solid phase extraction (SPE) method. The extracts were analyzed using LC–atmospheric pressure chemical ionization (APCI)–MS in the negative ionization mode. The limits of detection of the extraction method ranged from 6.8 to 3.2 ng mL⁻¹. Using the optimized method, 14 drinks and 3 tea products were analyzed. Concentrations of total marker compounds in drinks ranged between 1685 and 21.4 ng mL⁻¹ with individual ginkgo terpene trilactones being detected at ppb concentrations. Analysis of brewed tea products presented much higher total marker compound concentrations ranging from 8.12 and 16.6 μg mL⁻¹. Analytical results reproducibility data, and recovery of the SPE method are presented.     

Rapid and sensitive liquid chromatography-mass spectrometry method for determination of ropinirole in human plasma

A rapid and robust liquid chromatography–mass spectrometry (LC–MS/MS) method was developed for non-ergoline dopamine D₂-receptor agonist, ropinirole in human plasma using Es-citalopram oxalate as an internal standard. The method involves solid phase extraction from plasma, reversed-phase simple isocratic chromatographic conditions and mass spectrometric detection that enables a detection limit at picogram levels. The proposed method was validated with linear range of 20–1200 pg/ml. The extraction recoveries for ropinirole and internal standard were 90.45 and 65.42%, respectively. The R.S.D.% of intra-day and inter-day assay was lower than 15%. For its sensitivity and reliability, the proposed method is particularly suitable for pharmacokinetic studies.     

Composition analysis of two batches of polysorbate 60 using MS and NMR techniques

Batch variation in Tween 60 has shown to influence the rheological properties of semisolid emulsions. MS (LC–MS, GC–MS, MSⁿ) and NMR (¹³C, ¹H, ¹H COSY and HMBC) techniques were used to analyze and compare the composition of two batches of Tween 60 with particular emphasis on the number of POE groups and their distribution within the molecule. Acid and saponification values were also determined. The batches contained different proportions of components (sorbitan polyethoxylates, sorbitan monoester–diester-polyethoxylates and isosorbide monoester–diester-polyethoxylates). The number of POE groups were averaged over the four sites in sorbitan and the two sites in isosorbide molecules. The batches differed from each other in terms of (i) the POE sorbitan stearate/POE sorbitan palmitate ratios (batch 1, 3:2 and batch 2, 4:5), (ii) the ratio of sorbitans to isosorbides (batch 1, 2:3; batch 2, 7:13); and (iii) the acid values (batch 1, 3.1; batch 2, 0). It is concluded that liquid chromatography combined with electrospray mass spectrometry and ion trap separation is a useful tool for establishing the compositional profile of different batches of Tweens. ¹H NMR could provide a simple and rapid pharmacopoeial test for the ratio of sorbitan to isosorbide in Tweens.     

High sensitive analysis of rat serum bile acids by liquid chromatography/electrospray ionization tandem mass spectrometry

Sensitive liquid chromatography (LC)/electrospray ionization (ESI) tandem mass spectrometry (MS) can be used to analyze the bile acid composition of rat serum. This method can analyze eight common bile acids and their glycine and taurine conjugates in 100 μl rodent serum by gradient elution on a reversed-phase column using a mixture of 20 mM ammonium acetate buffer (pH 8.0), acetonitrile and methanol as a mobile phase. Selected reaction monitoring analysis under negative ion detection mode allowed the achievement of a high sensitive assay with a simple solid phase extraction using an ODS cartridge column. We used this method to investigate the effect of a one-day fast on the concentration and composition of serum bile acids in rats. The results suggested that the method described here is useful for the dynamic analysis of serum bile acids in rats.     

L-amino acid oxidase from Vipera lebetina venom: isolation, characterization, effects on platelets and bacteria.

The L-amino acid oxidase from Vipera lebetina venom was purified to homogeneity using combination of size exclusion, ion exchange and hydrophobic chromatography. The monomeric molecular mass of the homodimeric enzyme is 60.9kDa. The N-terminal and the tryptic peptides share high homology with other snake venom L-amino acid oxidases. The enzyme displays high specificity towards hydrophobic L-amino acids, the best substrates are L-Met, L-Trp, L-Leu followed by L-His, L-Phe, L-Arg and L-Ile. Six substrates-Gly, L-Ser, L-Thr, L-Pro, L-Cys, L-Asp--were not oxidized. The enzyme has antimicrobial activity inhibiting the growth of both Gram-negative and Gram-positive bacteria. V. lebetina LAAO dose-dependently inhibited platelet aggregation induced by ADP or collagen. In case of ADP-induced aggregation the inhibitory effect was more pronounced on the second wave of aggregation.     

Quantitative determination of total methamphetamine and active metabolites in rat tissue by liquid chromatography with tandem mass spectrometric detection

High-throughput liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methodology for the determination of methamphetamine (METH), amphetamine (AMP), 4-hydroxymethamphetamine (4-OH-METH), and 4-hydroxyamphetamine (4-OH-AMP) was developed and validated using simple trichloroacetic acid sample treatment. The method was validated in rat serum, brain, and testis. Lower limits-of-quantitation (LOQ) for METH and AMP were 1 ng x mL(-1) using positive ion electrospray tandem mass spectrometry (MS/MS). The accuracy of the method was within 25% of the actual values over a wide range of analyte concentrations. The within-assay precision was better than 12% (coefficient of variation). The method was linear over a wide dynamic range (0.3-1000 ng x mL(-1)). Quantitation was possible in all 3 matrices using only serum standards because of minimal matrix-associated ion effects or the use of an internal standard. Finally, the LC-MS/MS method was used to determine serum, brain, and testis METH and AMP concentrations during a subcutaneous infusion (5.6 mg kg(-1) day(-1)) of METH in rats. Concentrations of 4-OH-AMP and 4-OH-METH were below the LOQ in experimental samples. The bias introduced by using serum calibrators for the determination of METH and AMP concentrations in testis and brain was less than 8% and insignificant relative to the interanimal variability.     

Personal observations on the role of the lumbrical muscles in carpal tunnel syndrome

A case of carpal tunnel syndrome is presented in which an abnormally high origin of a lumbrical muscle seemed to be the cause. Since the patient and author are the same, the symptoms of compression and during the postoperative period are well documented. Evoking the symptoms by exercise or active motion is suggested for future diagnostic tests. A surgical incision more ulnarward is recommended.     

Determination of sodium monofluoroacetate (compound 1080) in biological tissues using oxygen combustion and a fluoride selective membrane electrode

Sodium monofluoroacetate (SMFA) was quantitated in plasma and liver homogenates after total fluoride analysis using an oxygen combustion method and fluoride selective electrode. The organic content of the samples were estimated by difference between total fluoride and inorganic fluoride. Recoveries (%±SD) of SMFA in aqueous samples, plasma and liver homogenates were 99.1 ± 2.3, 95.2 ± 3.3 and 95.3 ± 2.9, respectively. The within-run coefficient of variation in the 10.0-0.072 mg/ml (or mg/g) range for all samples was 4.7% or less. The method is suitable for quantitation of SMFA in biological materials and as a laboratory diagnostic aid in cases of acute SMFA intoxication.     

Comparison of the metabolic profiles of benzo[alpha]pyrene obtained from primary cell cultures and subcellular fractions derived from normal and methylcholanthrene-induced rat liver

Hepatocyte primary cell (HPC) cultures derived from either (a) non-induced (normal) or (b) methylcholanthrene (MC)-induced rat liver actively metabolized the carcinogen benzol[alpha]pyrene (BaP) over a 24-h period. In both cases, the BaP metabolites generated were qualitatively similar to those seen in the metabolism of BaP by isolated rat liver microsomal fractions; in addition, unidentified compounds were evident in the chromatographic profile generated by the cultured cells. In cells derived from (a), levels of known metabolites (phenols and diols) increased over the time period studied. On the other hand, in cells derived from (b), levels of diols decreased markedly after 8 h. These results suggest that induction with MC enhances both activation and, to a greater extent, conjugative-detoxification pathways of BaP, so that in cells obtained from (b) the formation of water-soluble metabolites is enhanced and levels of organic soluble metabolites are lower than in cells obtained from (a). Metabolism of BaP in primary cell culture derived from rat liver is thus seen to be similar to in vivo metabolism of the carcinogen, but somewhat in contrast to the in vitro microsomal (subcellular) metabolism of BaP where conjugative-detoxification pathways are virtually inoperative.