Ultrasound Assisted Extraction of Saponins from Hedera helix L. and an In Vitro Biocompatibility Evaluation of the Extracts.

The aim of this study was to establish the best ultrasound assisted extraction (UAE) conditions of saponins from Hedera helix L. leaves and to evaluate the in vitro biocompatibility of the extracts richest in saponins. Different parameters, such as extraction time, temperature, ultrasound power, solvent to plant material ratio, and solvent concentration, were investigated. The most efficient extraction conditions were a temperature of 50 °C, an ultrasound amplitude of 40%, an extraction time of 60 min, a plant material to solvent ratio of 1:20 (w:v), and 80% ethanol as solvent. In vitro cytotoxicity of the extracts richest in saponins and their influence on the DNA content of L929 (NCTC) fibroblasts were tested. Until 200 µg/mL, the studied extracts were cytocompatible with L929 fibroblast cell lines at 48 h of treatment. These in vitro cell culture results provide useful information for further applications of Hedera helix extracts in a pharmaceutical field.

New Pyrazolo-Benzimidazole Mannich Bases with Antimicrobial and Antibiofilm Activities.

A new series of pyrazolo-benzimidazole hybrid Mannich bases were synthesized, characterized by 1H-NMR, 13C-NMR, IR, UV-Vis, MS, and elemental analysis. In vitro cytotoxicity of the new compounds studied on fibroblast cells showed that the newly synthesized pyrazolo-benzimidazole hybrid derivatives were noncytotoxic until the concentration of 1 µM and two compounds presented a high degree of biocompatibility. The antibacterial and antibiofilm activity of the newly synthesized compounds was assayed on Gram-positive Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212, and Gram-negative Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922 strains. All synthesized compounds 5a-g are more active against all three tested bacterial strains Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212, and Escherichia coli ATCC25922 than reference drugs (Metronidazole, Nitrofurantoin), with the exception of compounds 5d and 5g, which are less active compared to Nitrofurantoin, and all synthesized compounds 5a-g are more active against Pseudomonas aeruginosa ATCC27853 compared to reference drugs (Metronidazole, Nitrofurantoin). Compound 5f showed the best activity against Staphylococcus aureus ATCC 25923, with a MIC of 150 µg/mL and has also inhibited the biofilm formed by all the bacterial strains, having an MBIC of 310 µg/mL compared to the reference drugs (Metronidazole, Nitrofurantoin).

A Fatty Acid Fraction Purified From Sea Buckthorn Seed Oil Has Regenerative Properties on Normal Skin Cells.

In recent years, natural product's research gained momentum, fueled by technological advancement and open availability of research data. To date, sea buckthorn (Hippophae rhamnoides L. [Elaeagnaceae]) plant parts, especially berries, are well characterized and repeatedly tested for antioxidant activity and regenerative properties, in various cell types and tissues. However, fatty acids (FA) have been less investigated in term of biological effects, although, they are important bioactive components of the sea buckthorn fruit and oil. The aim of our work was to determine whether sea buckthorn seed oil is a suitable source of FA with regenerative properties on normal skin cells. Using high-performance liquid chromatography (HPLC) and liquid chromatography - mass spectrometry (LC-MS), we purified and characterized four fractions enriched in saturated (palmitic) and non-saturated (linoleic, alfa-linolenic, oleic) FA, which were tested for cytotoxicity, cytokine and growth factor production, and regenerative effect on normal keratinocytes and skin fibroblasts. Evidence is presented that the palmitic acid enriched fraction was a suitable sea buckthorn seed oil derived product with cell proliferation properties on both skin cell types.

Sea-Buckthorn Seed Oil Induces Proliferation of both Normal and Dysplastic Keratinocytes in Basal Conditions and under UVA Irradiation.

Past decades demonstrate an increasing interest in herbal remedies in the public eye, with as many as 80% of people worldwide using these remedies as healthcare products, including those for skin health. Sea buckthorn and its derived products (oil; alcoholic extracts), rich in flavonoids and essential fatty acids, are among these healthcare products. Specifically, sea buckthorn and its derivatives are reported to have antioxidant and antitumor activity in dysplastic skin cells. On the other hand, evidence suggests that the alteration of lipid metabolism is related to increased malignant behavior. Given the paradoxical involvement of lipids in health and disease, we investigated how sea-buckthorn seed oil, rich in long-chain fatty acids, modifies the proliferation of normal and dysplastic skin cells in basal conditions, as well as under ultraviolet A (UVA) radiation. Using real-time analysis of normal and dysplastic human keratinocytes, we showed that sea-buckthorn seed oil stimulated the proliferation of dysplastic cells, while it also impaired the ability of both normal and dysplastic cells to migrate over a denuded area. Furthermore, UVA exposure increased the expression of CD36/SR-B2, a long-chain fatty acid translocator that is related to the metastatic behavior of tumor cells.

Monitoring Methylmalonic Aciduria by NMR Urinomics.

The paper reports on monitoring methylmalonic aciduria (MMA)-specific and non-specific metabolites via NMR urinomics. Five patients have been monitored over periods of time; things involved were diet, medication and occasional episodes of failing to comply with prescribed diets. An extended dataset of targeted metabolites is presented, and correlations with the type of MMA are underlined. A survey of previous NMR studies on MMA is also presented.

Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography-mass spectrometry and ion mobility mass spectrometry.

Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 µm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 µm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 µm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean C max of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.

Development of a sensitive method for simultaneous determination of fluticasone propionate and salmeterol in plasma samples by liquid chromatography-tandem mass spectrometry.

A new method for the fast simultaneous quantification of fluticasone propionate and salmeterol from plasma samples by liquid chromatography-tandem mass spectrometry, with adequate sensitivity for pharmacokinetic applications, was developed and validated. The chromatographic separation and mass-spectrometric parameters were optimized for the retention and detection of the two compounds, despite quite different structures and properties. Two columns connected in series were used, cation-exchange (Zorbax 300-SCX, 5 cm x 2.1 mm, 5 µm) and octadecyl (Discovery HSC18, 10 cm x 2.1 mm, 5 µm). The mass-spectrometric interface was operated in negative electrospray ionization mode; high sensitivity and lesser matrix effects were obtained, permitting smaller consumption of plasma. The sample preparation was based on supported liquid-liquid extraction in 96-well format plates that provided clean samples with a simplified procedure that was suitable for automation. The method was validated according to regulatory guidelines, by assessing lower limits of quantification, selectivity, linearity, accuracy, precision, extraction recoveries and matrix effects. A comparison with two other methods for the separate determination of fluticasone propionate and salmeterol in plasma samples, previously developed by our group, is presented. The statistical evaluation of the results obtained with the three methods on a set of unknown samples from treated patients demonstrated good correlation (R² 0.987 for fluticasone propionate and 0.967 for salmeterol).

Development and validation of an HPLC-MS/MS method to quantify clopidogrel acyl glucuronide, clopidogrel acid metabolite, and clopidogrel in plasma samples avoiding analyte back-conversion.

A new sensitive and fast quantitative analytical method for the simultaneous determination of clopidogrel, its main metabolite clopidogrel carboxylic acid, and the newly described acyl glucuronide metabolite, in human plasma samples, is presented. The analytical procedures (plasma storage, handling, and extract storage in the autosampler) were optimized in order to avoid back-conversion; a known drawback in measurements of clopidogrel. Clopidogrel acyl glucuronide was confirmed as a major source of back-conversion to the parent drug in the presence of methanol, and thorough stability experiments were carried out to find the most appropriate conditions for an accurate analysis of clopidogrel and the two metabolites. The method was validated by assessing selectivity, sensitivity, linearity, accuracy, and precision for all three analytes, in accordance to Food and Drug Administration guidelines. Spiked quality controls in plasma as well as incurred samples were used to verify back-conversion in the selected conditions, with results meeting European Medicines Agency acceptance criteria (concentrations within 80-120% of the first reading). The method was then applied to a pharmacokinetic study, and for the first time, a pharmacokinetic curve of clopidogrel acyl glucuronide in human plasma is presented. The concentrations ranged up to 1,048.684 ng/mL, with a mean of 470.268 ng/mL, while clopidogrel had a mean C(max) of 1.348 ng/mL; these orders of magnitude show how much the back-conversion of this metabolite may influence clopidogrel quantification if it is not properly controlled.

A one year investigation of the occurrence of illicit drugs in wastewater from Brussels, Belgium.

Daily 24 hour composite influent wastewater samples were collected from the wastewater treatment plant of Brussels-North (Belgium) for eight months to study variations in the concentrations and mass loads of nine illicit drugs and metabolites: cocaine (COC) and its metabolites benzoylecgonine (BE) and ecgonine methyl ester (EME), amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methamphetamine (METH), methadone (MTD) and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and the specific metabolite of heroin, 6-monoacetylmorphine (6-MAM). Samples were analyzed using a validated analytical method based on solid-phase extraction and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry. The selected compounds could be detected and quantified in all samples. The measured concentrations were in agreement with earlier published concentrations, when available. All illicit drugs showed significant differences in mass loads between months. Daily variations were observed for MDMA, AMP and COC and metabolites, with higher mass loads observed during the weekend. This is probably related to their recreational consumption pattern and was further confirmed when holiday periods (New Year's Eve, Belgian national holiday) were in detail investigated. For METH, 6-MAM, MTD and EDDP, stable and consistent amounts were observed during the week. In all samples, the ratio of the concentration of the parent compound to its metabolite(s) was calculated to evaluate whether measured concentrations reflect human excretion of the illicit drugs. For MTD, the ratio of parent compound/metabolite was in agreement with the excretion pattern, while for COC some deviations were observed, resulting from excretion pattern uncertainties, and stability and discharge issues.

Application of hydrophilic interaction chromatography for the analysis of polar contaminants in food and environmental samples.

For the analysis of highly hydrophilic and polar compounds, Hydrophilic Interaction Chromatography (HILIC) has been established as a valuable complementary approach to reversed-phase liquid chromatography (RPLC). Moreover, the use of mobile phases with a high percentage of organic solvent in HILIC separation is beneficial for mass spectrometric (MS) detection, because of enhanced ionization which results in an increased sensitivity. In this review, various applications of HILIC are described for a number of environmental and food contaminants together with detailed methodological descriptions and the advantages or drawbacks of HILIC compared to other LC methods are critically discussed. In the first part of the review, an overview is given of the work that has been carried out with HILIC for the analysis of pharmaceuticals and pesticides in environmental samples. HILIC has shown its applicability for polar pharmaceuticals, such as antibiotics, estrogens and their metabolites, drugs of abuse, cytostatics, metformin and contrast agents. In the pesticide group, HILIC chromatography was helpful for polar phenylurea and organophosphorus pesticides. The second part of the review focuses on the analysis of antibiotic residues in food and feed with HILIC, while in the pesticide group, HILIC experiments have been reported for dithiocarbamates and quaternary ammonium compounds. The last chapter gives an overview of the analysis by HILIC of miscellaneous analytes in aquatic and food/feed samples.

Sewage epidemiology--a real-time approach to estimate the consumption of illicit drugs in Brussels, Belgium.

The sewage epidemiology approach was applied to a one-year sampling campaign in the largest wastewater treatment plant (WWTP) in Belgium. The consumption of cocaine (COC), amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methamphetamine (METH), methadone (MTD) and heroin (HER) was evaluated based on measured concentrations of the parent compound and/or metabolites in daily 24-hour composite influent wastewater samples. The inevitable back-calculations used in the sewage epidemiology approach were adapted to newly available information regarding the stability of the compounds in wastewater and the excretion pattern of illicit drugs. For COC, three different back-calculation approaches were evaluated. In addition, for the first time, efforts were made to calculate the number of inhabitants living in the catchment area of the WWTP in a real-time and dynamic way, based on concentrations of nitrogen, phosphorus and oxygen in the wastewater samples. Clear variations in the amount of inhabitants in the catchment area of the WWTP were observed. For COC, AMP and MDMA a significant higher weekend use was observed while for HER and MTD no significant daily variations could be found. METH consumption was negligible. Generally, the sewage epidemiology calculations were in agreement with official statistics. This manuscript shows that sewage epidemiology provides consistent and logical results and that it is a promising tool that can be used in addition to classical studies to estimate illicit drug use in populations. Therefore, efforts should be made to further optimize this approach in the future.

Illicit drug consumption estimations derived from wastewater analysis: a critical review.

The consumption of illicit drugs causes indisputable societal and economic damage. Therefore it is necessary to know their usage levels and trends for undertaking targeted actions to reduce their use. Recently, a new approach (namely sewage epidemiology) was developed for the estimation of illicit drug use based on measurements of urinary excreted illicit drugs and their metabolites in untreated wastewater. This review aims at critically evaluating the published literature and identifying research gaps of sewage epidemiology. Firstly, the existing analytical procedures for the determination of the four most used classes of illicit drugs worldwide (cannabis, cocaine, opiates and amphetamine-like stimulants) and their metabolites in wastewater are summarized and discussed. The focus lies on the sample preparation and on the analysis with chromatographic techniques coupled to mass spectrometry. Secondly, back-calculations used to transform measured concentrations in wastewater (in ng/L) into an amount of used illicit drug (in g/day per 1000 inhabitants or doses/day per 1000 inhabitants) are discussed in detail for the four groups of illicit drugs. Sewage epidemiology data from Spain, Belgium, UK, Italy, Switzerland and USA are summarized and compared with data from international organisations, such as the European Monitoring Centre for Drug and Drug Addiction (EMCDDA) and the United Nations Office on Drugs and Crime (UNODC). The results derived from wastewater analysis show in general good agreement with existing prevalence data (percentage of a population that uses illicit drugs at a given time) and demonstrate the potential of sewage epidemiology. However, this review confirms that future work should focus on further optimisation and standardisation of various important parameters (e.g. sample collection and back-calculations). In the future, sewage epidemiology could be used in routine drug monitoring campaigns as a valuable tool in addition to the classical socio-epidemiological studies for the determination of local, national and international illicit drug use.

Simultaneous determination of 15 top-prescribed pharmaceuticals and their metabolites in influent wastewater by reversed-phase liquid chromatography coupled to tandem mass spectrometry.

A fast and sensitive high performance reversed-phase liquid chromatography-tandem mass spectrometry method was developed and validated for the analysis of 15 prescription pharmaceuticals and four of their metabolites in influent wastewater. The selected pharmaceuticals belonged to various classes, such as angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, calcium antagonists, ß-blockers, antidepressants, analgetics, anticonvulsants, platelet antiaggregants, and cholesterol lowering agents. They were selected from the list of top-sold prescription pharmaceuticals in Belgium. The chromatographic separation was optimized in order to achieve suitable retention times, good resolution for analytes susceptible of mass spectrometric cross-talk and high sensitivity in one single run. All compounds eluted within 9 min on a Phenomenex Kinetex C18 column, based on a newly developed technology that allows a very narrow distribution of the core-shell particles, providing high separation efficiency. Sample preparation was executed with solid-phase extraction on Oasis MCX cartridges. The method was validated by assessing specificity, selectivity, lower limit of quantification (LLOQ), linearity, accuracy, precision, extraction recovery, and matrix effects following Food and Drug Administration guidelines. The method LLOQs ranged from 0.5 to 25 ng/L. Calibration curves and LLOQs were designed to provide a good analytical performance at concentrations expected in real influent wastewater samples for each target compound. Eight deuterated analogues were used as internal standards for quantification. The method was applied to influent wastewater samples collected from 17 different wastewater treatment plants throughout Belgium. Most of the analytes were measured in the samples at concentrations above LLOQ. Seven of the compounds were for the first time reported in influent wastewater. The newly developed analytical method is currently used to assess relationships between sales figures of pharmaceuticals and their corresponding concentrations in influent wastewater.

Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma. Use of incurred samples to test back-conversion.

Quantitative methods using LC-MS/MS allow achievement of adequate sensitivity for pharmacokinetic studies with clopidogrel; three such methods, with LLOQs as low as 5 pg/mL, were developed and fully validated according to the well established FDA 2001 guidelines. The chromatographic separations were performed on reversed phase columns Ascentis RP-Amide (15 cm x 2.1 mm, 5 µm), Ascentis Express C8 (10 cm x 2.1 mm, 2.7 µm) and Ascentis Express RP Amide (10 cm x 2.1 mm, 2.7 µm), respectively. Positive electrospray ionization in MRM mode was employed for the detection and a deuterated analogue (d3-clopidogrel) was used as internal standard. Linearity, precision, extraction recovery, matrix effects and stability tests on blank plasma spiked with clopidogrel and stored in different conditions met the acceptance criteria. During the analysis of the real samples from the first pharmacokinetic study, a significant increase (>100%) of the measured clopidogrel concentrations in the extracts kept in the autosampler at 10 °C was observed. Investigations led to the conclusion that most probably a back-conversion of one or more of the clopidogrel metabolites is occurring. The next methods were optimized in order to minimize this back-conversion. After a series of experiments, the adjustment of the sample preparation (e.g. processing at low temperature and introducing a clean-up step on Supelco HybridSPE-Precipitation cartridges) has proven to be the most effective in order to improve the stability of the extracts. Incurred samples of real subjects were successfully used in the validation of the last two analytical methods to evaluate the back-conversion, while tests using only the known metabolites could not detect this important problem.

Optimization and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the determination of 13 top-prescribed pharmaceuticals in influent wastewater.

A sensitive hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry method was developed and validated for the analysis of 13 pharmaceuticals (omeprazole, pantoprazole, ranitidine, citalopram, fluoxetine, paroxetine, venlafaxine, tramadol, nebivolol, metoprolol, atenolol, bisoprolol and metformin) in influent wastewater. The analytes were selected from the list of top-sold prescription pharmaceuticals in Belgium. The HILIC separation was optimised to achieve quantification of all analytes in real influent wastewater samples where other compounds, mainly metabolites of some of the pharmaceuticals, were found to interfere even with mass-spectrometric detection in multiple reaction monitoring (MRM) mode. Sample cleanup and preconcentration was based on solid-phase extraction, and Oasis HLB cartridges were chosen after optimization. The method was validated by assessing the following parameters: specificity, selectivity, lower limit of quantification (LLOQ), linearity, accuracy, precision, recovery and matrix effects. For each analyte, LLOQs were sufficiently low to provide a good analytical performance at concentrations expected in real influent samples. Typical LLOQs were 1 ng/L, except for metformin (500 ng/L). Six deuterated analogues were used as internal standards. A total of 22 influent wastewater samples collected from 18 different wastewater treatment plants in Belgium were analysed. Most analytes were present in the samples at concentrations above the LLOQ and were in agreement with other results from the literature. Nebivolol was for the first time found in influent wastewater. In the future, this analytical method will be used to determine if there is a relationship between pharmaceutical sales figures and concentrations of these compounds in influent wastewater.

Ethyl glucuronide determination in meconium and hair by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Ethyl glucuronide (EtG) detection in non-conventional matrices, such as hair and meconium, can provide useful information on alcohol abuse over a long time frame, for example during pregnancy or after a withdrawal treatment. This study reports on the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the analysis of EtG in meconium and hair. For each matrix, the sample preparation and the chromatographic separation were thoroughly optimised. Additionally, experiments with reversed-phase liquid chromatography were also performed in the development stages. Analyses were carried out using a Phenomenex Luna HILIC column (150 mm x 3 mm, 5 microm) and a mobile phase composed by ammonium acetate 2mM and acetonitrile, in gradient. Different SPE cartridges (Oasis MAX, Oasis WAX, aminopropyl silica) and solvents were tested in order to obtain the highest recoveries and cleanest extracts. Optimal results were obtained for meconium with aminopropyl cartridges, while for hair an incubation of 16 h with 2 mL of water and acetonitrile (50/50, v/v) provided good results. The analytical method was validated for both matrices (meconium and hair) by assessing linearity, precision, accuracy, recovery and limit of quantification. The calibration curve concentrations ranged from 50 to 1200 pg/mg for meconium and from 20 to 1000 pg/mg for hair. Real meconium and hair samples were analyzed and results were consistent with literature.

Analysis of drugs of abuse in wastewater by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The simultaneous analysis of nine drugs of abuse (DOAs) and their metabolites (amphetamine, methamphetamine, methylenedioxymethamphetamine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, cocaine, benzoylecgonine, ecgonine methyl ester and 6-monoacetylmorphine) in wastewater based on hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) was optimised and validated. For each analyte, the deuterated analogue was used for quantification. The separation by HILIC showed good performance for all compounds, especially for the hydrophilic compounds, which elute early (amphetamine-like stimulants) or show no retention (ecgonine methyl ester) in reversed-phase liquid chromatography. Sample preparation based on solid-phase extraction was optimised by comparing Oasis HLB and Oasis MCX sorbents for various parameters such as sample pH, amount of sorbent bed and washing solvent. The method was validated for each compound by assessing the following parameters (following International Conference on Harmonisation guidelines): specificity, limit of quantification (LOQ), linearity, accuracy, precision, recovery and matrix effects. LOQs were 2 ng/L for 6-monoacetylmorphine, ecgonine methyl ester and amphetamine and 1 ng/L for the rest of the compounds, corresponding with the lowest point in the calibration curve. Except for 6-monoacetylmorphine, all compounds were detected from 1 to 819 ng/L in influent wastewater samples (n = 12) collected from 11 different wastewater treatment plants across Belgium. The presence of ecgonine methyl ester in wastewater could be demonstrated for the first time. In the future, the new HILIC-MS/MS method will be applied to assess the use of DOAs in Belgium using the "sewage epidemiology" approach.

High-throughput HPLC-MS/MS method to determine ibandronate in human plasma for pharmacokinetic applications.

A sensitive high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of ibandronate in human plasma. In a previous study, we have analyzed alendronate in urine samples of subjects treated at therapeutic dosages, using a derivatization approach; a similar derivatization was adapted and improved to determine ibandronate in plasma. The bisphosphonate was isolated from the biological matrix by liquid-liquid extraction, and derivatized with trimethylsilyldiazomethane prior to separation on a reversed-phase column (Supelco Discovery HSC18) and detection on a quadrupole-linear ion trap mass spectrometer (API 4000 QTrap). Various parameters of extraction and derivatization were optimized in order to get adequate recovery, high derivatization yield and minimal ion suppression; a deuterated analogue, d3-ibandronate, was used as internal standard. The transitions 376.1-->114.2 and 379.1-->61.0 were acquired to monitor ibandronate and d3-ibandronate derivatives, respectively. A multiplexing LC system made possible the overlapping of two chromatographic runs, thus the interval between injections being reduced to only 2min, a very short analysis time for compounds of this class. The method was fully validated over the quantification range 0.2-175.0ng/ml, allowing an appropriate evaluation of the plasma concentrations of ibandronate, expected at therapeutic dosage, as proved by application to a pharmacokinetic study. A good linearity over the selected range (r>0.99), accuracy and precision within +/-15% of the target values and a recovery over 50% were obtained.

Development and application of a high-performance liquid chromatography-mass spectrometry method to determine alendronate in human urine.

Despite the high potential offered by electrospray ionization on highly polar compounds like biphosphonates, few applications have been developed. High-performance liquid chromatography (HPLC) separation methods suitable for such molecules cannot be used in tandem with mass spectrometry (MS) due to high non-volatile salt content; at the same time the sample preparation, in biological fluids, is also a challenging problem. In the past ion-pair chromatography was mainly used in the case of HPLC-MS of biphosphonates, but no application to quantitative pharmacokinetic (PK) studies has been presented. In this study, after preliminary tests with ion-pair chromatography showing a poor sensitivity, a combined derivatization of the amino group and the biphosphonate has been developed and tested in a PK study. Using this analytical approach we were able to fully validate the quantitation of alendronate in the range of 6.667-4860.0 ng/ml in urine (sample volume 2.0 ml); each analytical run was 5.0 min long. The sensitivity achieved permitted a correct evaluation of the alendronate urinary excretion over the full period of urine collection. Sample preparation despite its complexity permitted to process and analyze up to 200 samples in a working day.