Validated UPLC-MS/MS method for the analysis of vitamin B6 pyridoxal 5́-phosphate, pyridoxal, pyridoxine, pyridoxamine, and pyridoxic acid in human cerebrospinal fluid.

Vitamin B6 and its metabolites play a crucial role in the development and interaction of brain metabolism. Following diagnostic improvements additional inherited disorders in vitamin B6 metabolism have been identified, most of them leading to a severe epileptic disorder accompanied by progressive neurological deficits including intellectual disability and microcephaly. Since early treatment can improve the outcome, fast and reliable detection of metabolic biomarkers is important. Therefore, the analysis of vitamin B6 metabolites has become increasingly important, but is, however, still challenging and limited to a few specialized laboratories. Until today, vitamin B6 metabolites are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using trichloroacetic acid for protein precipitation. In this work, we present the development and validation of a new, accurate and reliable method for analysis and quantification of the vitamin B6 vitamers pyridoxal 5́-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and pyridoxic acid (PA) in human CSF samples using acetonitrile for protein precipitation. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (UPLC-ESI-MS/MS). The calibration was performed in surrogate matrix Ringer solution and metabolites were quantified by their corresponding isotopically labelled internal standards. A protein precipitation by acetonitrile was applied greatly improving chromatographic separation of the metabolites in a 4.7 min chromatographic run. The method was validated following the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The metabolites were quantified from 5 to 200 nmol/L with a seven-point calibration curve and minimum coefficient of regression of 0.99. The validation was performed with quality control samples at four concentration levels with surrogate matrix ringer solution and pooled CSF material. Within- and inter-day accuracy and precision in Ringer solution were within 85.4 % (PLP) and 114.5 % (PM) and from 2.6 % (PA) to 16.5 % (PLP). Within- and inter-day accuracy and precision in pooled CSF material were within 90.5 % (PN) and 120.1 % (PL) and from 1.7 % (PA) to 19.0 % (PM). The method was tested by measuring of 158 CSF samples to determine reference ranges. The B6 vitamers PLP and PL were determined in all CSF samples above 5 nmol/L while PN, PM and PA showed concentrations below or near LOQ. Probable supplementation of PLP was detected in eight CSF samples, which revealed high concentrations of PM, PN, PL, or PA, whereas PLP was in the reference range or slightly elevated. The method is suitable for the application within a routine diagnostic laboratory.

Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the quantification of the antidiabetic drug metformin and six others pharmaceuticals in wastewater.

A rapid and sensitive hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry method was developed and fully validated for the simultaneous determination of the antidiabetic drug metformin and six further pharmaceuticals (atenolol, gabapentin, levofloxacin, ciprofloxacin, propranolol and trimethoprim) in influent and effluent wastewater. Five deuterated related compounds were used as internal standards in order to control the extraction, injection and ionization variability. Two solid phase extraction methods and Oasis HLB 1cc cartridge, using only 1mL sample with acid or basic pH-value were optimized and compared in order to match the specificity of both influent and effluent wastewater matrixes. The most important challenge was to efficiently extract the polar compound metformin from the aqueous matrix. This was possible by adjusting the sample pH to 10. On the other hand, the adjustment to acid pH prevents metformin binding on the SPE cartridges. Metformin was so directly recovered after passing through the cartridge and injected in the HPLC-MS/MS system without any other preparation. These two methods present advantages and drawbacks, but are both applicable to wastewater samples. The method with extraction at pH 3 was applied to influent wastewater samples and metformin concentrations higher than 625ng/L. On the other hand, the extraction method at pH 10 with a lower limit of quantification for metformin of 156ng/L was successfully applied to effluent water and to surface water samples. Both methods were fully validated. Limits of quantification were sufficiently low to provide good analytical performances for the determination of all drugs in both influent and effluent wastewater. Wastewater samples collected from six different wastewater treatment plants in Germany were analyzed. All analytes were present in influent samples at concentrations above the lower limit of quantification LLOQ, particularly metformin which presents concentrations up to 300µg/L.

Post-column infusion of internal standard quantification for liquid chromatography-electrospray ionization-tandem mass spectrometry analysis - Pharmaceuticals in urine as example approach.

Liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) technique is gaining more and more attraction as the method of choice for multi-sample analysis. However, it is strongly susceptible to the influence of matrix components. Matrix effects are the main source of substantial losses in detection sensitivity and have to be compensated via complex quantification methods In this work, we introduce a sophisticated quantification method for the LC-ESI-MS/MS analysis of 16 substances in urine samples using a single continuously post-column infused internal standard (PCI-IS) for matrix effect correction. The performance of the introduced technique was proven by the simultaneous quantification using internal standards. Our results demonstrate that a single post-column infused internal standard suffices to analyze multiple target analytes. The introduced method is a new approach to analyze complex matrices and represents a powerful alternative to the classic internal standard methodology. The proposed technique significantly reduces the required steps for sample preparation, costs of additional stable isotopically-labeled internal standards, and self-induced matrix effects.

Evaluation of the matrix effect of different sample matrices for 33 pharmaceuticals by post-column infusion.

Matrix effects that occur during quantitative measurement by liquid chromatography mass spectrometry specifically when using electrospray ionization are a widely recognized phenomenon. Sample matrix compounds affect the ionization process of the target analytes, lead to a low signal response, and flawed analytical results. How these matrix compounds directly influence the ionization process has not yet been completely understood. In the present study, we determined the matrix effect for 33 pharmaceutical substances in sample extracts of urine, plasma and wastewater. Most of the investigated substances were subject to a signal suppression effect. Only for a small subset of the compounds we detected a signal enhancement effect. We investigated the matrix effect profiles in detail to disentangle the influence of different matrices and to correlate the impact of specific components and groups of the analyzed extract in suppressing or enhancing effects in the profile. Most signal suppression effects were detected in the first half of the chromatographic run-time for the matrix extracts of urine and wastewater. The observed effects are caused by high mass flow of salts and other diverse matrix components that were contained in high concentrations in those biological matrices. We also found signal suppression in the matrix effect profile of plasma samples over a wide time range during the chromatographic separation that were associated with a high content of triglycerides of diverse carbohydrate chain lengths. Here, we provide a broader picture of how 33 substances were influenced during analysis. Our results imply that a high number of the investigated substances had comparable effects of matrix compounds, despite differences in their chemical structure.

Development of a SPE-HPLC-MS/MS method for the determination of most prescribed pharmaceuticals and related metabolites in urban sewage samples.

Based on regional prescription data several pharmaceuticals with variable amounts of prescription and corresponding metabolites were selected and analyzed in influent and effluent samples of the sewage treatment plant (STP) in Dresden, Germany. Pharmaceuticals of the following most prescribed therapeutic groups were chosen: antibiotics, antifungals, anticonvulsants, antipsychotics, antidepressants, and cardiovascular active compounds like beta blockers and angiotensin-converting enzyme inhibitors. To analyze the selected compounds, a multi-target method was developed and applied to 24-h composite wastewater samples for three single days in May and June 2014. The method was based on a cleanup of a sample with a volume of 1mL using solid phase extraction followed by a high performance liquid chromatography coupled to a tandem mass spectrometer. Analytes were separated in a 15min chromatographic separation and quantified using 23 Internal Standards and a calibration curve in 40-fold diluted blank urine. The limit of quantification varied between 50 and 200ng/L and for all analytes good accuracy and precision as well as linearity for the calibration curve with the correlation coefficient R(2) higher than 0.99 was reached. A total of 41 and 40 of the selected 55 analytes were detected and quantified in the influent and effluent samples of the studied STP, respectively. Valsartan was the compound with the highest maximum concentration in influent (27.1µg/L) and effluent (15.7µg/L). Furthermore, analytes like bezafibrate, candesartan, carbamazepine, gabapentin, metoprolol, levetiracetam, pregabalin and telmisartan as well as the metabolite O-desmethyl venlafaxine were detectable in influent and effluent samples, respectively, with a concentration higher than 1µg/L.

Simultaneous determination of most prescribed antibiotics in multiple urban wastewater by SPE-LC-MS/MS.

A rapid analytical method was developed for the application of a long-term monitoring (>one year) of the most prescribed and often in hospitals used antibiotics in diverse wastewaters of an urban sewage treatment plant (STP). Additionally to the selected multi-class antibiotics amoxicillin, penicillin V and piperacillin (penicillins), cefotaxime and cefuroxime (cephalosporins), azithromycin, clarithromycin and roxithromycin (macrolids), ciprofloxacin and levofloxacin-ofloxacin (fluoroquinolones), clindamycin (lincosamide), doxycycline (tetracycline), sulfamethoxazole (sulfonamide) and trimethoprim (dihydrofolate reductase inhibitor), the bioactive metabolite clindamycin-sulfoxide, the reserve antibiotic vancomycin (glycopeptide) and as tracer of the STP the anticonvulsant carbamazepine and the antifungal fluconazole were involved. The analytical method combines a low-sample-volume solid phase extraction (SPE), followed by a chromatographic separation using a reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) technique, respectively, coupled to a triple quadrupole mass spectrometer. Detection was performed with multiple reaction monitoring (MRM) measured with positive electrospray ionization (ESI+). The extraction efficiency of different SPE cartridges and optimized pH-values of the preparation procedure were tested. Finally, the extraction of antibiotics was realized with the Oasis HLB cartridge and a pH adjustment at 3.5. An external calibration curve in diluted blank urine was used for quality control of the sample set of daily composite samples of the STP for the duration of one year monitoring. The squared coefficient of determination (r(2)) in the concentration range (20-20,000ng/L or 100-100,000ng/L) of the calibration curves for the method was higher than 0.99 for all determined substances. The limit of quantification (LoQ) ranged between 0.8ng/L (azithromycin) and 245.1ng/L (vancomycin). Furthermore, a standard addition was used for quantification in wastewater samples. The process efficiencies ranged from 20% (doxycycline) to 134% (cefuroxime) in influent samples and from 31% (doxycycline) to 171% (cefuroxime) in effluent samples of the STP. All selected substances have been found in wastewater samples. Cefuroxime, doxycycline, levofloxacin, piperacillin, sulfamethoxazole and carbamazepine showed highest concentrations up to 6.2µg/L.