Development and validation of a simple HPLC-MS/MS method for the quantification of methylmalonic acid in human serum without a derivatization step.

A simple and rapid HPLC-MS/MS analytical method was developed and validated for the determination of methylmalonic acid (MMA) in human serum without a derivatization step. Serum samples (200 µl) were pretreated using a simple method based on ultrafiltration using a VIVASPIN 500 ultrafiltration column. Chromatographic separation was achieved on a Luna Omega C18 column with a PS C18 precolumn guard by gradient elution using 0.1% (v/v) formic acid in water (mobile phase A) and 0.5% (v/v) formic acid in acetonitrile (mobile phase B) at a flow rate of 0.2 ml min-1 . The total run time of the analysis was 4.5 min. Negative electrospray ionization and multiple reaction monitoring mode were used. The lower limit of detection and lower limit of quantification for MMA were determined to be 13.6 and 42.3 nmol L-1 , respectively. The developed method enabled the quantification of MMA in a wide linear range of 42.3-4230 nmol L-1 with a correlation coefficient of 0.9991.

A new HPLC-MS/MS analytical method for quantification of tazobactam, piperacillin, and meropenem in human plasma.

A simple and fast high-performance liquid chromatography with tandem mass spectrometry method for quantification of tazobactam, piperacillin, and meropenem in human plasma has been developed and validated. Simple sample preparation with a volume of 10 µL was done by protein precipitation with a mixture of methanol-acetonitrile-water (6:2:2, v/v/v). Chromatographic separation was achieved on a Luna column with a precolumn security guard by gradient elution using a mobile phase consisting of water with the addition of 0.1% formic acid (component A) and mixture methanol-acetonitrile (8:2, v/v) with the addition of 0.1% formic acid (component B). The run time was 2.7 min. The lower limits of detection and lower limits of quantification were for piperacillin 0.03 and 0.1 mg/L, for meropenem 0.04 and 0.2 mg/L and for tazobactam 0.16 and 0.5 mg/L. The validated method was used for therapeutic monitoring of tazobactam, piperacillin, and meropenem in samples of patients treated in the intensive care unit.

A new HPLC-MS/MS method for simultaneous determination of Cyclosporine A, Tacrolimus, Sirolimus and Everolimus for routine therapeutic drug monitoring.

A rapid, simple and robust HPLC-MS/MS method for simultaneous determination of immunosuppressants Cyclosporine A, Tacrolimus, Sirolimus and Everolimus has been developed and validated. Sample of whole blood with volume of 50 µL was prepared by a protein precipitation with methanol and 0.5 mol. L-1 ZnSO4. Chromatographic separation was achieved on a Phenyl-Hexyl column by a gradient elution using 20 mmol.L-1 ammonium formate/0.1% (v/v) formic acid in water (mobile phase A) and 20 mmol.L-1 ammonium formate/0.1% (v/v) formic acid in methanol (mobile phase B) with flow rate 1 mL.min-1. The run time was 3.5 min. Electrospray ionization and multiple reaction monitoring was used. The lower limit of quantification (LLOQ) was set at 0.5 µg.L-1 for Tacrolimus, Sirolimus and Everolimus and 5 µg.L-1 for Cyclosporine A. The method demonstrated adequate accuracy and precision with sufficient linearity range.

Human metabolism and excretion kinetics of the fragrance 7-hydroxycitronellal after a single oral or dermal dosage.

7-Hydroxy-3,7-dimethyl-1-octanal, also known as 7-hydroxycitronellal (7-HC, CAS No. 107-75-5) is a synthetic fragrance widely used in cosmetic and hygiene products. Due to its large scope, 7-HC was selected for the development of a biomonitoring method suitable for the general population within the frame of the cooperation project between the German Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI). In a human study with 5 healthy subjects who received single dermal and oral doses 7-HC, suitable metabolites and their urinary excretion kinetics was investigated. Two metabolites of 7-hydroxycitronellal were identified in urinary fractions after dermal and oral dosing: The alcohol 7-hydroxycitronellol (7-HCO) and the corresponding acid 7-hydroxycitronellylic acid (7-HCA). Only 7-HCA proved to be a suitable biomarker of exposure to 7-HC, since 7-HCO was quantifiable in only a minority of urine samples collected from the general population. Quantification of 7-HCA was conducted by means of a newly developed UPLC-MS/MS (ultra-high pressure liquid chromatography combined with tandem mass spectrometry) method. Peak excretion of 7-HCA occurred between 3 and 5h after oral application and about 10h after dermal administration. Due to the limited skin absorption of 7-HC, 7-HCA concentrations after dermal application were much lower than levels after oral application. After 24h, about 9% and 50% of the dermally and orally applied dose, respectively, were excreted as 7- HCA. With the conversion factors derived from the controlled human study, we estimated median exposure doses in a group of 40 human volunteers from the general population of approximately 93µg 7-HC per day. In conclusion, the 7-HC metabolite 7-HCA in urine is a suitable biomarker of exposure and can be applied for biomonitoring of the general population.

A validated UPLC-MS/MS method for biomonitoring the exposure to the fragrance 7-hydroxycitronellal.

7-Hydroxycitronellal is a synthetic fragrance (CAS No. 107-75-5) which is used commonly in cosmetics, washing- and cleaning agents and as flavoring in foods. Due to its broad application in various fields, 7-hydroxycitronellal was selected for the development of a biomonitoring method for the quantitative exposure assessment within the frame of the cooperation project of the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI). For this purpose, an ultra performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) based method was developed for the determination of potential biomarkers of 7-hydroxycitronellal (7-HC) in human urine samples. 7-Hydroxycitronellylic acid (7-HCA) turned out to be the quantitatively most important metabolite of 7-HC in human urine, occurring in 1000 times higher amounts than 7-hydroxycitronellol (7-HCO) or other potential metabolites. Therefore, an analytical method for 7-HCA was developed using stable isotope-labeled 7-HCA as internal standard (IS). The method includes a cleavage step of possible metabolite conjugates with an enzyme mix of ß-glucuronidase and arylsulfatase. Subsequent sample cleanup was performed by liquid-liquid extraction (LLE) with dichloromethane. The method was calibrated by calculating the linear regression between the analyte/IS ratio and the nominal 7-HCA concentrations in water. The method was validated according to approved standard guidelines and proved to be robust, reliable and sensitive for the human biomonitoring of 7-HC. The method was applied to urine samples of 40 adult volunteers from the general population. 7-HCA was quantifiable in urine of all subjects. Thus the developed method proved to be suitable for assessing the background exposure to 7-HC in the general population.

Human metabolism and excretion kinetics of the fragrance lysmeral after a single oral dosage.

2-(4-tert-Butylbenzyl)propionaldehyde, also known as lysmeral, lilial or lily-aldehyde (CAS No 80-54-6) is a synthetic fragrance used in a variety of consumer products like perfumes, after shave lotions, cosmetics and others. Due to its broad application, lysmeral was selected for the development of a biomonitoring method for the general population within the frame of the cooperation project of the Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI). The project also comprises the identification of suitable biomarkers of exposure in human urine as well as basic toxicokinetic data after defined, experimental exposure. For this purpose, 5 healthy subjects were orally dosed once with 5.26mg lysmeral. Urine was collected immediately before and for 48h after administration of the fragrance. The lysmeral metabolites lysmerol, lysmerylic acid, hydroxylated lysmerylic acid and 4-tert-butylbenzoic acid (TBBA) were determined in all urine samples by a newly developed UPLC-MS/MS (ultra-high pressure liquid chromatography combined with tandem mass spectrometry) method. Peak excretion for all metabolites occurred between 2 and 5h after oral application, with the primary metabolites (lysmerol and lysmerylic acid) being excreted about 1h earlier than the secondary metabolites (hydroxylated lysmerylic acid and TBBA). More than 90% of all measured lysmeral metabolites were excreted after 12h, with the renal excretion being virtually complete after 48h. After this time period, TBBA, lysmerol, lysmerylic acid and hydroxyl-lysmerylic acid represent on average 14.3, 1.82, 0.20 and 0.16%, respectively, of the dose administered. In total, the 4 metabolites determined represent about 16.5% of the dose. With the conversion factors derived from the controlled human study, we estimated median exposure doses for lysmeral in a group of 40 human volunteers from the general population of approximately 140-220µg per day. In conclusion, the lysmeral metabolites lysmerol, lysmerylic acid, hydroxyl-lysmerylic acid and TBBA in urine are suitable biomarkers of exposure and can be applied, either single or in any combination, for biomonitoring of the general population.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the human biomonitoring of non-occupational exposure to the fragrance 2-(4-tert-butylbenzyl)propionaldehyde (lysmeral).

2-(4-tert-Butylbenzyl)propionaldehyde also known as lysmeral, lilial, or lily aldehyde (CAS No. 80-54-6) is a synthetic odorant mainly used as a fragrance in a variety of consumer products like cleaning agents, fine fragrances, cosmetics, and air fresheners. Due to its broad application in various fields, lysmeral was selected for the development of a biomonitoring method for the quantitative exposure assessment within the frame of the cooperation project of the Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI). A method based on ultra-high pressure liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of potential biomarkers of lysmeral in human urine samples. Sample cleanup was performed by liquid-liquid extraction (LLE). Quantification was achieved by standard addition using stable isotope-labeled, authentic reference standards. The method is characterized by its robustness, reliability, and excellent sensitivity as proven during method validation according to approved standard guidelines. The following five lysmeral metabolites were identified as potential biomarkers of exposure for lysmeral in human urine samples: lysmerol, lysmerylic acid, hydroxylated lysmerylic acid, tert-butylbenzoic acid (TBBA), and tert-butylhippuric acid (TBHA). The determination of lysmerol required derivatization with 3-nitrophthalic acid anhydride and showed the lowest limit of detection (LOD) and limit of quantification (LOQ) in urine (0.035 and 0.10 µg/L, respectively). LOD and LOQ for the other metabolites were in the range of 0.12-0.15 and 0.36-0.45 µg/L, respectively. Accuracy for all analytes was in the range of 90-110 %. Intra- and inter-day precision was in the range of 5-10 %, except for TBHA, for which the coefficient of variation was unacceptably high (>20 %) and therefore excluded from the method. The method was applied to urine samples of 40 adult volunteers. The four remaining lysmeral metabolites were detectable in most of the 40 urine samples in the following order according to quantity excreted: TBBA >> lysmerol ≈ lysmerylic acid > hydroxy-lysmerylic acid. In conclusion, we successfully developed a biomonitoring method for the assessment of the exposure to lysmeral in the general population. The method is characterized by its precision, robustness, and accuracy. The metabolites lysmerol, lysmerylic acid, hydroxylated lysmerylic acid, and TBBA turned out to be suitable biomarkers of exposure to lysmeral, either alone or in combination with one or more of the other metabolites. Sensitivity was found to be sufficient for assessing the background exposure to this chemical in the general population.