Quantitative NMR (qNMR) spectroscopy based investigation of the absolute content, stability and isomerization of 25-hydroxyvitamin D2/D3 and 24(R),25-dihydroxyvitamin D2 in solution phase.

Vitamin D is an important parameter, in serum/plasma based diagnostic analysis, for the determination of optimal regulation of calcium and phosphate homeostases in the human body, vital for the monitoring/progression of osteomalacia and rickets. Particularly, the quantification of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D in blood is an excellent indicator for the vitamin D status of a patient. For this purpose, LC-MS/MS methods, based on appropriate vitamin D reference standards, are considered to be 'gold standard' for such measurements. We have utilized quantitative NMR spectroscopy to determine the absolute content of these molecules, available as non-certified chemicals, and have determined the stability of these callibrators in borderline polar solvents at room temperature. We have observed significant isomerization of the analytes, which can play a big role in quantification of these analytes by hyphenated LC and GC analytical techniques. Appropriate explanations are given for the observation of new impurities with time in solution phase. The spin system selected for quantitation was determined using relevant 1D and 2D NMR pulse sequences. The advantage of the qNMR approach is that it is based on the quantification of atoms rather than molecular properties (e.g., quantitation by LC/UV, GC, etc.). Since the signals in an NMR spectrum are different nuclear spin-systems dispersed precisely in a magnetic environment, with the intensity being directly proportional to the amount of a particular type of nuclear spin, this technique delivers unparalleled information about the chemical structure and the absolute content.

Absolute content determination by quantitative NMR (qNMR) spectroscopy: a curious case of aldosterone.

Quantitative NMR spectroscopy has been utilized to calculate the absolute content (g g-1) of aldosterone, which is necessary for electrolyte balance and blood pressure regulation, in commercially available materials. Explanations have been provided for many signals observed in the 1HNMR spectrum, false interpretation of which can have significant effects if such a value is utilized for the primary calibrators in ID-LC-MS/MS ('gold standard') reference methods in clinical chemistry.

An isotope dilution LC-MS/MS-based candidate reference method for the quantification of androstenedione in human serum and plasma.

The accurate measurement of androstenedione in human serum and plasma is required for steroid profiling to assure the appropriate diagnosis and differential diagnosis of hyperandrogenism. In this work, we introduce an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) candidate reference measurement procedure for the quantification of androstenedione in human serum and plasma. The performance of the procedure enables its use in the evaluation and standardization of routine assays and for the evaluation of patient samples to ensure the traceability of individual patient results. As the primary standard, a certified reference material from NMIA (National Measurement Institute, Australia) was used. Additionally, a quantitative nuclear magnetic resonance (qNMR) method was developed for the value assignment of the primary reference material, which ensures the direct traceability to SI units, as well as the independence from the availability of reference materials. 13C3-labeled androstenedione was used as the internal standard. The introduced method allows the measurement of androstenedione in the range of 0.05-12 ng/mL, and the assay imprecision was found to be <2% between 5 and 12 ng/mL, 3.5% at 1.5 ng/mL, and 5.2% at 0.05 ng/mL, with an accuracy of 95-105% for the serum and 91-103% for the plasma matrix. The transferability to a second laboratory was validated by method comparison based on 112 patient samples. The comparison of the results obtained from the presented method and an LC-MS/MS routine assay, using 150 native patient samples, showed a good correlation with a bias of the routine method of ≤4.0%.

An isotope dilution LC-MS/MS based candidate reference method for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood.

An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and 13C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus (k = 2).

An LC-MS/MS based candidate reference method for the quantification of carbamazepine in human serum.

A validated LC-MS/MS-based candidate reference measurement procedure for the quantification of carbamazepine is presented in order to be used for standardization and harmonization of routine assays applied for therapeutic drug monitoring. Sample preparation was based on protein precipitation using acetonitrile followed by sample dilution. Since the previously listed certified reference material (CRM) SRM 1599 (anticonvulsant drug level assay standard) is no longer available, an ISO certified calibration material was used in this assay. As internal standards deuterated analyte congeners were applied. The method allows the measurement of carbamazepine, carbamazepine-10,11-epoxide and 10-hydroxy-10,11-dihydrocarbamazepine in the concentration range of 0.1 to 22.0µg/ml with LODs and LOQs of <0.1µg/ml and 0.1µg/ml, respectively. Comparative measurement of 105 native patient samples using the here presented method showed a good agreement between two independent laboratories with a mean bias of 0.6%.

An LC-MS/MS based candidate reference method for the quantification of total gentamicin in human serum and plasma using NMR characterized calibrator material.

BACKGROUND: Accurate measurement of gentamicin concentration in serum and plasma is required for therapeutic drug monitoring to ensure appropriate treatment of patients. In this work, we present a validated LC-MS/MS-based candidate reference measurement procedure for total gentamicin quantification to be used for standardization and harmonization of routine assays applied for therapeutic drug monitoring of this compound. Total gentamicin is the sum of the concentrations of five known congeners C1, C1a, C2, C2a and C2b. To our knowledge, there is so far no LC-MS method for quantification of total gentamicin in human serum described in literature. METHODS: Sample preparation was based on sample dilution with an aqueous internal standard solution followed by protein precipitation. Stable derivatives of gentamicin-glycine congeners were prepared by chemical synthesis and used as internal standards. The primary calibration material used in this assay was characterized by NMR spectroscopy and the pattern of the gentamicin congeners was determined. The total gentamicin was reported as the sum of the congeners which were quantified individually by LC-MS/MS. RESULTS: The method allows the measurement of total gentamicin in human serum and plasma in the concentration range of 0.1 to 12.0µg/ml with an assay imprecision of ≤6% CV and an assay accuracy between 96% and 114%. LOD and LOQ for the total gentamicin were 0.04µg/ml and 0.13µg/ml, respectively. Comparative measurement of 128 native patient samples using this method implemented at two laboratory sites showed an excellent agreement. CONCLUSIONS: Validation results proved that this protocol describes a robust and reliable method which is suggested as reference measurement procedure for the standardization and harmonization of routine assays for the quantification of total gentamicin.

Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling.

OBJECTIVES: For quantification of 25-hydroxyvitamin D(3) (25OH-D(3)), 25-hydroxyvitamin D(2) (25OH-D(2)), 3-epi-25-hydroxyvitamin D(3) (3-epi-25OH-D(3)) and 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)-D(3)) in human serum a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated. DESIGN AND METHODS: After protein precipitation further purification is achieved with on-line sample preparation using a reversed phase (RP) C-4 column. Chromatographic separation is realized by a RP-column with core shell material and pentafluorophenyl (PFP) selectivity. Atmospheric pressure chemical ionization in the positive ion mode with multi-reaction monitoring is used for analyte detection. RESULTS: Baseline separation of the analytes is achieved below 10 min. The method is linear over the range 4.0-265.3 nmol/L for 25OH-D(3), 3.9-183.6 nmol/L for 25OH-D(2), 2.0-133.8 nmol/L for 3-epi-25OH-D(3) and 2.8-129.9 nmol/L for 24R,25(OH)(2)-D(3) (r(2)>0.998). The limit of quantification is 4.0 nmol/L for 25OH-D(3), 3.9 nmol/L for 25OH-D(2), 2.0 nmol/L for 3-epi-25OH-D(3) and 2.8 nmol/L for 24R,25(OH)(2)-D(3). The CVs for the intra-day and inter-day precision are <5% and <4%, respectively. Metabolite levels for a set of 50 human serum samples have been determined and resulted in the detection of considerable amounts of 3-epi-25OH-D(3) and 24R,25(OH)(2)-D(3). CONCLUSIONS: This highly specific HPLC-MS/MS method is suitable for vitamin D profiling. There is a correlation between 25OH-D(3) and 24R,25(OH)(2)-D(3). Serum concentration of 24R,25(OH)(2)-D(3) increases disproportionally with increasing concentration of 25OH-D(3).