A newly developed and validated LC-MS/MS method for measuring 7-dehydrocholesterol (7DHC) concentration in human skin: a tool for vitamin D photobiology research.

BACKGROUND: UVB absorption by 7-dehydrocholesterol (7DHC) in the skin triggers the production of vitamin D and its metabolites, which maintain calcium homeostasis. Detection and measurement of 7DHC in skin using modern liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques have been lacking, yet there is need for such a technique to provide more information on 7DHC concentration and its UVB responses in human skin. OBJECTIVES: To develop and validate a reliable method to measure 7DHC concentration in skin. METHODS: Human skin punch biopsies of 5 mm diameter obtained through the Manchester Skin Health Biobank were utilised. 7DHC was extracted with ethyl acetate:methanol 1:1 (v/v) and derivatised using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), to allow for improved ionisation of 7DHC through Electrospray Ionisation Mass Spectrometry (ESI-MS). Solid supported liquid extraction (SLE) was also employed to allow the removal of larger lipids from 7DHC and minimise potential matrix effects. RESULTS: The LC-MS/MS assay satisfied International Council for Harmonisation research standards for method validation. Calibration curve was linear with a typical r2 of 0.997, coefficient of variation was 11.1% and 4.32% for inter-assay and intra-assay imprecision, respectively. Lower limit of quantification was 1.6 µg/g and upper limit of quantification was 100 µg/g, SLE recovery of 7DHC was on average 91.4%. CONCLUSIONS: We have developed a robust, precise and accurate assay for the detection and quantification of 7DHC in small samples of human skin (0.2 cm2 surface area). This novel method of extraction and quantification will be valuable to future vitamin D photobiology research.

Vitamin D Measurement, the Debates Continue, New Analytes Have Emerged, Developments Have Variable Outcomes.

The demand for measurement of vitamin D metabolites for clinical diagnosis and to advance our understanding of the role of vitamin D in human health has significantly increased in the last decade. New developments in technologies employed have enabled the separation and quantification of additional metabolites and interferences. Also, developments of immunoassays have changed the landscape. Programmes and materials for assay standardisation, harmonisation and the expansion of the vitamin D external quality assurance scheme (DEQAS) with the provision of target values as measured by a reference measurement procedure have improved standardisation, quality assurance and comparability of measurements. In this article, we describe developments in the measurement of the commonly analysed vitamin D metabolites in clinical and research practice. We describe current analytical approaches, discuss differences between assays, their origin, and how these may be influenced by physiological and experimental conditions. The value of measuring metabolites beyond 25 hydroxyvitamin D (25(OH)D), the marker of vitamin D status, in routine clinical practice is not yet confirmed. Here we provide an overview of the value and application of the measurement of 1,25 dihydroxyvitamin D, 24,25 dihydroxyvitamin D and free 25OHD in the diagnosis of patients with abnormalities in vitamin D metabolism and for research purposes.

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment.

BACKGROUND: Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. METHODS: Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. FINDINGS: A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. CONCLUSIONS: In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. TRIAL REGISTRATION NUMBER: EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

Comparative Study of Healthy Older and Younger Adults Shows They Have the Same Skin Concentration of Vitamin D3 Precursor, 7-Dehydrocholesterol, and Similar Response to UVR.

Vitamin D3 synthesis in human skin is initiated by solar ultraviolet radiation (UVR) exposure of precursor 7-dehydrocholesterol (7DHC), but influence of age on the early stage of vitamin D3 metabolism is uncertain. We performed a prospective standardised study in healthy ambulant adults aged ≥65 and ≤40 years examining (1) if baseline skin 7DHC concentration differs between younger and older adults and (2) the impact of older age on serum vitamin D3 response to solar simulated UVR. Eleven younger (18-40 years) and 10 older (65-89 years) adults, phototype I-III, received low-dose UVR (95% UVA, 5% UVB, 1.3 SED) to ~35% of the body surface area. Biopsies were taken for 7DHC assay from unexposed skin, skin immediately and 24 h post-UVR, and blood sampled at baseline, 24 h and 7 d post-UVR for vitamin D3 assay. Samples were analysed by HPLC-MS/MS. Baseline skin 7DHC (mean ± SD) was 0.22 ± 0.07 and 0.25 ± 0.08 µg/mg in younger versus older adults (no significant difference). Baseline serum vitamin D3 concentration was 1.5 ± 1.5 and 1.5 ± 1.7 nmol/L in younger versus older adults, respectively, and showed a significant increase in both groups post-UVR (no significant differences between age groups). Thus, skin 7DHC concentration was not a limiting factor for vitamin D3 production in older relative to younger adults. This information assists public health guidance on sun exposure/vitamin D nutrition, with particular relevance to the growing populations of healthy ambulant adults ≥65 years.

Reference intervals for serum 24,25-dihydroxyvitamin D and the ratio with 25-hydroxyvitamin D established using a newly developed LC-MS/MS method.

24,25(OH)2D is the product of 25(OH)D catabolism by CYP24A1. The measurement of serum 24,25(OH)2D concentration may serve as an indicator of vitamin D catabolic status and the relative ratio with 25(OH)D can be used to identify patients with inactivating mutations in CYP24A1. We describe a LC-MS/MS method to determine: (1) the relationships between serum 24,25(OH)2D and 25(OH)D; (2) serum reference intervals in healthy individuals; (3) the diagnostic accuracy of 24,25(OH)2D measurement as an indicator for vitamin D status; 4) 24,25(OH)2D cut-off value for clinically significant change between inadequate and sufficient 25(OH)D status. Serum samples of healthy participants (n=1996) from Army recruits and patients (n=294) were analysed. The LC-MS/MS assay satisfied industry standards for method validation. We found a positive, concentration-dependent relationship between serum 24,25(OH)2D and 25(OH)2D concentrations. The 25(OH)D:24,25(OH)2D ratio was significantly higher (P<.001) at 25(OH)D<50 nmol/L. The reference interval for 25(OH)D:24,25(OH)2D ratio in healthy subjects was 7-23. Measurement of serum 24,25(OH)2D can be used as predictor of vitamin D status, a concentration of>4.2 nmol/L was identified as a diagnostic cut-off for 25(OH)D replete status. One patient sample with an elevated 25(OH)D:24,25(OH)2D ratio of 32 and hypercalcaemia who on genetic testing confirmed to have a biallelic mutation of CYP24A1. Our study demonstrated the feasibility of a combined 24,25(OH)2D and 25(OH)D assessment profile. Our established cut-off value for 24,25(OH)2D and ratio reference ranges can be useful to clinicians in the investigation of patients with an impaired calcium/phosphate metabolism and may point towards the existence of CYP24A1 gene abnormalities.

Serum markers in alkaptonuria: simultaneous analysis of homogentisic acid, tyrosine and nitisinone by liquid chromatography tandem mass spectrometry.

BACKGROUND: Alkaptonuria is a rare debilitating autosomal recessive disorder of tyrosine metabolism, where deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid. Homogentisic acid is deposited as an ochronotic pigment in connective tissues, especially cartilage, leading to a severe early onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, nitisinone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of homogentisic acid. METHOD: A reverse phase liquid chromatography tandem mass spectrometry method has been developed to simultaneously analyse serum homogentisic acid, tyrosine and nitisinone. Using matrix-matched calibration standards, two product ion transitions were identified for each compound (homogentisic acid, tyrosine, nitisinone) and their respective isotopically labelled internal standards ((13)C6-homogentisic acid, d2-tyrosine, (13)C6-nitisinone). RESULTS: Intrabatch accuracy was 94-108% for homogentisic acid, 95-109% for tyrosine and 89-106% for nitisinone; interbatch accuracy (n = 20) was 88-108% for homogentisic acid, 91-104% for tyrosine and 88-103% for nitisinone. Precision, both intra- and interbatch were <12% for homogentisic acid and tyrosine, and <10% for nitisinone. Matrix effects observed with acidified serum were normalized by the internal standard (<10% coefficient of variation). Homogentisic acid, tyrosine and nitisinone proved stable after 24 h at room temp, three freeze-thaw cycles and 24 h at 4℃. The assay was linear to 500µmol/L homogentisic acid, 2000µmol/L tyrosine and 10µmol/L nitisinone; increased range was not required for clinical samples and no carryover was observed. CONCLUSIONS: The method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of alkaptonuria patients, pre- and post-nitisinone therapy.

Measurement of teicoplanin by liquid chromatography-tandem mass spectrometry: development of a novel method.

BACKGROUND: Teicoplanin is an antibiotic used for the treatment of endocarditis, osteomyelitis, septic arthritis and methicillin-resistant Staphylococcus aureus. Teicoplanin is emerging as a suitable alternative antibiotic to vancomycin, where their trough serum levels are monitored by immunoassay routinely. This is the first report detailing the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring teicoplanin in patients' serum. METHODS: An Acquity™ UPLC (ultra-pressure liquid chromatography) tandem mass spectrometer was used to measure teicoplanin concentrations in samples from patients, quality assurance schemes and quality control preparations. Ristocetin was successfully implemented as a suitable internal standard. Ion suppression, linearity, stability, matrix effects, recovery, imprecision, lower limits of quantification and detection, interference and method comparison against immunoassay were all assessed. RESULTS: Teicoplanin and ristocetin had elution times of 1.39 and 1.24 min, respectively. Ion suppression was shown to be negligible, and linear calibration curves (0-200 µg/mL) were consistently reproduced to have r(2) values >0.99. Postextraction stability was achieved up to 20 h, while matrix effects were minimal coupled with sample recovery of >93%. The lower limit of quantification was 1 µg/mL, and 0.2 µg/mL was the lower limit of detection. Interference with other antibiotics was dependent on the combination of drugs present in patients' serum. A method comparison between immunoassay and LC-MS/MS suggested a negative bias for tandem mass spectrometry. CONCLUSIONS: This novel method of teicoplanin determination by LC-MS/MS is proven to be a robust protocol that is consistent and reproducible. Clinicians searching for alternatives in therapeutic drug monitoring may have an additional option that is potentially more accurate and specific.