Liquid chromatography-atmospheric pressure chemical ionisation/mass spectrometry: a rapid and selective method for the quantitative determination of ginkgolides and bilobalide in ginkgo leaf extracts and phytopharmaceuticals

In order to evaluate the composition of active constituents in phytopharmaceutical preparations, valid analytical methods are required. For the determination of the active terpene constituents of Ginkgo biloba (the ginkgolides and bilobalide), a liquid chromatography-mass spectrometry (LC-MS) method has been developed using atmospheric pressure chemical ionisation (APCI) in the negative ion mode. This detection mode was found to be much more sensitive and selective compared to UV; indeed the ginkgo terpene trilactones lack strong UV chromophores and flavonoids interfere with their UV detection. LC-APCI/MS detection allowed a considerable reduction in analysis time when compared to LC-UV, because LC resolution was only needed between the pair of isomers ginkgolide B and ginkgolide J. All compounds were selectively detected by single ion monitoring of their specific deprotonated molecules [M-H]-. The samples were directly injected without pre-purification, and a fast gradient was applied, reducing the total time of analysis to 14 min. With this method, the ginkgo terpene trilactones were detected on-line in the picogram range. Several commercial ginkgo preparations on the Swiss market were analysed, and the ginkgolide and bilobalide contents were evaluated using the method described.     

Central effects of yohimbine on copulatory behavior in aged male rats

It is well known that yohimbine has a history of popular use because of its supposed aphrodisiac properties. The present study was done to determine whether yohimbine can modify the copulatory behavior of aged male rats. Adult male rats of the Wistar-Imamichi strain, 52 weeks of age and weighing 600-650g, were injected intracerebroventricularly with yohimbine hydrochloride (5, 10 micrograms/10 microliters/rat) or vehicle. Each male was then given the opportunity to mate with a receptive female for 30 min after administration of yohimbine or vehicle. Yohimbine produced significant decreases in the latency to initial mounting and significant increases in the number of mountings. However, there was no ejaculation in the yohimbine-and vehicle-treated males. This study is the first to clearly establish an important modulator of sexual arousal for yohimbine in aged male rats.     

Liquid chromatographic tandem mass spectrometric determination of five coccidiostats in poultry eggs and feed

A method is described which permits the quantitative detection of the chemical coccidiostats halofuginone, robenidine, diclazuril, nicarbazin and dimetridazole and its main metabolite 2-hydroxydimetridazole in poultry eggs and feed. Sample preparations were kept very simple and are based upon extraction with an organic solvent. Sample extracts were injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system on a C18 column and a gradient elution was performed. Dimetridazole-D3 and diclazuril-bis, a structural analogue of diclazuril, were used as internal standards. Detection was performed on a triple quadrupole mass spectrometer in the selected reaction monitoring mode after ionisation in the positive or negative electrospray ionisation mode. Argon was applied as collision gas for collision induced dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC [Official Journal of the European Communities L221 (2002) 8].     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.     

Fluorescence of neurotransmitters and their reception in plant cell

The use of fluorescent reagents for the histochemical detection of catecholamines or histamine, as well as luminescent antagonists of the intracellular neurotransmitters revealed that they can bind to certain cellular compartments. After the treatment with glyoxylic acid (a reagent used for the detection of catecholamines), blue fluorescence with maximum at 460–475 nm was visualized in nuclei and chloroplasts (in control preparations no emission in this spectral region was recorded), as well as an intense fluorescence, exceeding the control level, in the vacuoles. After the exposure to ortho-phthalic aldehyde (a reagent used for the histamine detection), blue emission was more noticeable in nuclei and chloroplasts, which correlates with previously observed effects on intact cells, such as pollen and vegetative microspores. A comparison of the intensities of the biogenic amine-related emission in various organelles showed that the greatest emission was in vacuoles and the weakest, in chloroplasts. Thus, on the surface, and possibly within the organelles, fluorescence could demonstrate the presence of biogenic amines. Antagonists of the neurotransmitters (dtubocurarine for acetylcholine; yohimbine for dopamine; norepinephrine and inmecarb for serotonin), which fluoresce in the blue and blue-green region and usually bind with the plasmalemma of intact cells, also interacted with the membranes of the organelles studied. Fluorescence intensity depended on the object; most prominent it was for yohimbine in the outer membrane of the nucleus, vacuoles, and chloroplasts.     

Aphrodisiac Use Associated with HIV Infection in Elderly Male Clients of Low-Cost Commercial Sex Venues in Guangxi,China: A Matched Case-Control Study

Background

Rising HIV infection rates have been observed among elderly people in Guangxi, China. Inexpensive aphrodisiacs are available for purchase in suburban and rural areas. This study aims to investigate the association between aphrodisiac use and increased HIV risk for middle-aged and elderly men in Guangxi.

Methods

A matched case-control study of aphrodisiac use-associated HIV infection was performed among male subjects over 50 years old who were clients of low-cost commercial sex venues in Guangxi. The cases were defined as clients who were HIV-positive and two controls were selected for each case. The cases and the controls were matched on the visited sex venue, age (±3 years), number of years of purchasing sex (±3 years), and educational attainment. Subjects were interviewed and tested for HIV. Paired t-test or McNemar Chi-squared test were used to compare the characteristics between the cases and controls. A stepwise conditional logistic regression was used to identify risk factors associated with HIV infection.

Findings

This study enrolled 103 cases and 206 controls. Aphrodisiac use (P = 0.02, odds ratio (OR) = 1.81, 95% CI = 1.08–3.04), never using condom during commercial sex encounter (P = 0.03, odds ratio (OR) = 1.82, 95% CI = 1.08–3.07), and lacking a stable partner (P = 0.03, odds ratio (OR) = 1.76, 95% CI = 1.05–2.98) were found to be risk factors for HIV infection among the study groups. For subjects reporting aphrodisiac use, the frequency of purchasing sex was positively correlated with the frequency of aphrodisiac use (r = 0.3; p = 0.02).

Conclusions

Aphrodisiac use was significantly associated with increased HIV infection risk in men over 50 years old who purchased commercial sex in the suburban and rural areas of Guangxi. Further research and interventions should address the links between aphrodisiac use, commercial sex work, condom use, and increased HIV transmission.     

Analysis of aristolochic acids and analogues in medicinal plants and their commercial products by HPLC-PAD-ESI/MS

An HPLC-UV-MS method for the analysis of aristolochic acids A, B, C and D, 7-OH-aristolochic acid A, and aristolic acid in a number of plant materials and their commercial products has been developed. HPLC with photodiode array detection and electrospray ionisation-MS in the selected ion monitoring mode allowed the identification of the target compounds and increased the selectivity of complex analyses such as those associated with multi-botanical preparations. The presented method was used to analyse 10 plant samples and six commercial products that possibly contained aristolochic acids. The resulting chromatographic profiles of the samples were significantly different from each other, and the method was directly transferred to HPLC-MS, which was used to confirm the presence of the six aristolochic acids mentioned above.     

High-performance liquid chromatography-coordination ion spray-mass spectrometry (HPLC-CIS/MS): a new tool for the analysis of non-polar compound classes in plant extracts using the example of Piper methysticum Forst

An efficient method to characterise complex plant extracts is described using the example of Piper methysticum Forst. (kava; Piperaceae). The method is based on the on-line coupling of high-performance liquid chromatography to a new detection technique: coordination ion spray-mass spectrometry (CIS/MS). CIS/MS is a universal, novel ionisation technique improving selectivity as well as sensitivity. Charged complexes were formed through addition of central complexing ions such as sodium, silver and cobalt. The advantages of CIS/MS detection compared with the electrospray ionisation detection are discussed. The experimental set-up and the application of this simple and robust technique is described to show the its various fields of application in the analysis of plant extracts.     

Identification of various lipolytic enzymes in crude porcine pancreatic lipase preparations using covalent fluorescent inhibitors

We developed a specific method for determination and discrimination of lipo-/estero-lytic enzymes in crude lipase preparations. Here we study the composition of commercial porcine pancreatic lipase (PPL), since it is widely used for bioconversions of synthetic and natural substrates. Our method is based on incubation of enzyme samples with fluorescently labeled alkyl- or dialkylglyceryl-phosphonates in an appropriate solvent followed by protein separation by electrophoresis and fluorescence detection with a CCD camera. After incubation with short-chain alkylphosphonate solubilized by taurodeoxycholate, crude PPL preparations showed a very weak band at 50 kDa, which is indicative of low PPL concentrations in these samples. In addition, seven other fluorescent bands were detected. The band at the lowest molecular weight corresponded to alpha-chymotrypsin. Two intensive fluorescent bands were in the molecular weight range of chymotrypsinogen (26 kDa) and four weak bands were in the range 20-24 kDa. Long-chain dialkylglycerophosphonate labeled two protein bands in crude PPL: alpha-chymotrypsin and a very intensive band corresponding to the molecular weight of chymotrypsinogen. Detection of cholesterol esterase (98 kDa) in crude PPL preparations depended on addition of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to the incubation mix, as demonstrated by spiking with cholesterol esterase. Thus, commercial crude PPL preparations contain a variety of estero-/lipo-lytic enzymes in addition to rather low amounts of active PPL, which should be considered when using crude PPL for bioconversions. Our method can also be used to show whether an isolated esterolytic activity corresponds to a single protein or isoenzymes. Here we confirm by 2D-electrophoretic separation of "pure" PPL that PPL exists as isoenzymes in different glycosylated forms.     

Trophoblastic beta-globulin in immunoglobulin preparations

The content of trophoblastic beta-globulin in 142 lots of commercial immunoglobulin preparations from 20 manufacturers, produced from placental, abortion and donor blood sera, has been studied. 83% of lots from abortion blood serum and 94% of lots from placental blood serum have been found to contain the admixture of this beta-globulin, its concentration in the lots from placental blood serum being significantly higher. The method for the detection of trophoblastic beta-globulin may be used for evaluating the quality of immunoglobulin preparations as it indicates the degree of their purification from placental proteins.     

Purification and analysis of a proteinaceous aphrodisiac pheromone from hamster vaginal discharge

Hormonally regulated proteinaceous material secreted in hamster vaginal discharge is detected via the vomeronasal organ and elicits copulatory behavior in males. The major soluble protein in estrous vaginal discharge has been isolated, characterized by molecular weight and amino acid content, and shown to have substantial aphrodisiac activity. The aphrodisiac activity of the purified protein is abolished by heating or proteolysis, and the native protein retains the activity after procedures for removing possible ligands such as volatile odorants, steroids, and peptides. This evidence that the protein is a reproductive pheromone indicates that the mammalian vomeronasal organ can mediate sensory detection of behaviorally relevant macromolecules.     

Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.     

Detection of hydroxyethylstarch (HES) in human urine by liquid chromatography-mass spectrometry

The objective of this study was to establish the possibility of using liquid chromatography coupled to mass spectrometry for the detection of hydroxyethylstarch (a corn starch derived product) in urine as an alternative to the current time consuming GC-MS methods. Analyses were performed using an ion trap instrument after acidic hydrolysis. Ionization was carried out using atmospheric pressure chemical ionisation (APCI) operated in negative ionization mode and detection was performed using MS(2). The results indicate that the developed method can successfully be applied as a fast and reliable method for the detection and identification of hydroxyethylstarch.     

Immunoenzyme method in the diagnosis of meningococcal infections

The materials on the development and use of the test system, based on the enzyme-linked immunosorbent assay (ELISA) and intended for the detection of specific group A and C meningococcal polysaccharides and type b Haemophilus influenzae polysaccharide in the spinal fluid of patients, are presented. In this work commercial preparations manufactured in the USSR were used, and all parameters of the assay were developed on the basis of these preparations. The study was made on the samples of spinal fluid from 410 patients; of these, 203 had meningococcal infection, 57 had purulent bacterial meningitides and 150 had other diseases (acute respiratory diseases, influenza, etc.). As demonstrated by the results of this study, ELISA proved to be a highly specific and sensitive technique. In the investigation of the spinal fluid samples from the patients with meningococcal infection the use of ELISA with bacteriological techniques increased the number of positive results to 67%; with countercurrent electrophoresis, to 78%; and with bacterioscopy, to 83.8%. ELISA is recommended for practical use as an auxiliary laboratory technique and as a rapid method for the diagnosis of meningococcal infection.     

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells

The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mmx2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs<15%. MPA was stable at room temperature in the autosampler over 48 h and at -20 degrees C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.     

Rapid screening method for determination of Ecstasy and amphetamines in urine samples using gas chromatography-chemical ionisation mass spectrometry

The need for analytical screening tests more reliable and valid to detect amphetamine and related "designer drugs" in biological samples is becoming critical, due to the increasing diffusion of these drugs on the European illegal market. The most common screening procedures based on immunoassays suffer a number of limitations, including low sensitivity, lack of specificity and limited number of detectable substances. This paper describes a screening method based on gas-chromatography-mass-spectrometry (GC/MS) using positive chemical ionisation (PCI) detection. Methanol was used as reactant gas in the ionisation chamber. Molecular ions of different compounds were monitored, allowing a sensitivity of 5-10 ng/ml with high selectivity. The sensitivity of the method gives positive results in samples taken 48-72 h after intake of one dose of 50-100 mg. The method is simple and rapid. Sample preparation was limited to one liquid-liquid extraction, without any hydrolysis and derivatisation. Hydrolysis is critical to identify metabolites excreted as conjugates. Blank urine samples spiked with known amounts of amphetamine (AM), methylamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) were analysed. The method was successfully tested on real samples of urine from people, whose use of amphetamine was suspected, and results were compared with results obtained with immunoassays.     

Determination of L-carnitine by flow injection analysis with NADH fluorescence detection

A flow injection analysis method for determining L-carnitine is reported. The system uses the enzyme L-carnitine dehydrogenase covalently immobilized to Eupergit C. The NADH produced by the action of the enzyme, which is proportional to the L-carnitine concentration, is quantified using fluorescence detection. The system response was rapid and had a wide range of linearity. At a flow rate of 0.2 ml/min, a detection limit of 1 microM (20 pmol) was obtained for L-carnitine, peak areas were linear up to 100 microM, and samples could be injected every 4 min. The method performed well as a routine assay, showing high sensitivity (54,000 AU/microM), a precision of 0.96%, and the ability to carry out 144 consecutive assays with an RSD of 1.47% (good stability). Comparisons were made with other known methods for L-carnitine determination. Presence of D-carnitine had no effect on L-carnitine assay. The analysis was valid for determining L-carnitine concentrations in commercial pharmaceutical preparations.     

In vivo real-time monitoring of aphrodisiac pheromone release of small white cabbage butterflies (Pieris rapae)

The study of insect behavior is of practical importance for developing possible control methods in Integrated Pest Management. Currently, one model of butterfly mating behavior suggests that the initial location of potential mates occurs visually followed by the release of one or more short-range male aphrodisiac pheromones. This model is supported by data obtained from field observations and inferences based on the behavioral effects of chemicals extracted or isolated using indirect and offline techniques. In this study, we performed in vivo real-time monitoring of the male aphrodisiac pheromones released by the small white cabbage male butterfly (Pieris rapae Linnaeus) using confined direct analysis in real time (cDART) mass spectrometry. cDART is a new method easily adapted to the study in real time of chemicals released into the environment by virtually any insect. The major compound released by the male Pieris rapae was identified as ferrulactone. The experimental results reported here indicate that the release of ferrulactone occurs less than 1 s after the male visualizes its partner, and reaches a maximum after about one half minute. This study is the first reported in vivo detection and monitoring of butterfly male aphrodisiac pheromones in real time.     

Spectrofluorimetric method for the determination of sulpiride in pharmaceutical preparations and human plasma through derivatization with 2‐cyanoacetamide

A sensitive and accurate spectrofluorimetric method has been developed for the determination of sulpiride in pharmaceutical preparations and human plasma. The developed method is based on the derivatization reaction of 2‐cyanoacetamide with sulpiride in 30% ammonical solution. The fluorescent derivatized reaction product exhibited maximum fluorescence intensity at 379 nm after excitation at 330 nm. The optimum conditions for derivatization reactions were studied and the fluorescence intensity versus concentration plot was found to be linear over the concentration range 0.2–20.0 µg/mL with a correlation coefficient of 0.9985. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.82 and 2.73 ng/mL, respectively. The proposed method was validated according to ICH guidelines. The effects of common excipients and co‐administered drugs were also studied. The accuracy of the method was checked using the standard addition method and percent recoveries were found to be in the range of 99.00–101.25% for pharmaceutical preparations and 97.00–97.80% for spiked human plasma. The method was successfully applied to commercial formulations and the results obtained for the proposed method were compared with a high‐performance liquid chromatography reference method and statistically evaluated using the Student's t‐test for accuracy and the variance ratio F‐test for precision. A reaction pathway was also proposed. Copyright © 2012 John Wiley & Sons, Ltd.