SUPPLEMENTARY MATERIAL

MATERIALS and METHODS

CHEMICAL REAGENTS

Pyridoxamine dihydrochloride, pyridoxal hydrochloride, pyridoxine hydrochloride, pyridoxamine 5-phosphate, pyridoxal 5-phosphate mono-hydrate and 4-pyridoxic acid were purchased from Sigma Aldrich (Gillingham, Dorset, UK).

Four deuterated vitamers were used as internal standards; Pyridoxal methyl D3 hydrochloride [>98% atom %D] was purchased from Isotec, pyridoxine D2 hydrochloride (5-hydroxymethyl-D2) [>98% atom %D] from CDN Isotopes, 4-Pyridoxic acid D2 (5,5’-D2) [>98%] was purchased from buchem BV and D2 Pyridoxal 5’-Phosphate was kindly supplied as a gift by Professor Coburn, Department of Chemistry, Indiana University, Purdue University, Forte Wayne.

HPLC grade methanol was purchased from Fisher Scientific. Acetic acid, heptafluorobutyric acid (HFBA) and trichloroacetic acid (TCA) were purchased from Sigma Aldrich.

Stock solutions of B6 vitamers, PA and the deuterated internal standards were made with purified deionised water and stored at -80°C. They were placed on ice and protected from the light during laboratory handling.

All vitamer calibrators were checked for the presence of other B6 vitamers and deuterated internal standards were analysed to check for the presence of the non-deuterated species. No appreciable amounts were detected using the method described in this paper, making them suitable for use in method development.

SAMPLE COLLECTION & PREPARATION

Pooled plasma for calibration, recovery and imprecision studies was collected from healthy adults not taking any medications or vitamin supplementation. Clinical plasma samples for diagnostic and follow up work presented here were obtained with consent under an ethically approved project from patients attending Great Ormond Street Hospital for Children, London.

Venous blood samples were taken into EDTA tubes (Sarstedt Monovette®) and were centrifuged at 3000 rpm for 10 minutes at 4 °C within 60 minutes of collection and the plasma removed immediately. Plasma samples were then stored at -80 °C until further use.

At the time of analysis, plasma samples were defrosted on ice and protected from light. Proteins were precipitated by mixing 60 μL of plasma with an equal volume of 0.6 N TCA diluted with water containing deuterated internal standards to produce a final TCA concentration of 0.3 N. The sample was vortexed thoroughly for 30 seconds, left on ice in the dark for 60 minutes and finally centrifuged at 10,000 rpm for 10 minutes at 4 °C. The resulting supernatant was transferred to a HPLC vial and placed in an autosampler where the samples were kept at 4 °C and protected from light. Previous work has shown B6 vitamers to be stable under these conditions (Midttun, Hustad, Solheim et al 2005).

LIQUID CHROMATOGRAPHY

A Waters Alliance 2795 LC system was used for delivery of the mobile phase. A HS F5 column (Supelco; 10 cm x 2.1 mm; 3 μm) fitted with a HS F5 guard column (2 cm x 2.1 mm; 5 μm) were used for analysis. These were kept at a constant temperature of 30 °C during the analysis. The flow rate was 0.2 ml/minute.

For analysis of B6 vitamers in plasma, the mobile phase consisted of the following three components: (A) 100% methanol; (B) 3.7% acetic acid; (C) 3.7% acetic acid containing 100 mM HFBA. This acidic mobile phase with an ion pairing agent is suited to the detection of highly polarised compounds such as B6 vitamers. The best chromatographic separation and peak shapes were achieved with the gradient profile shown in Table 1. Each gradient step was linear. A chromatogram is shown in Figure 1.

ELECTROSPRAY IONISATION-TANDEM MASS SPECTROMETRY (ESI-MS/MS)

Mass spectrometry of B6 vitamers and pyridoxic acid was carried out using a triple quadrupole Micro Quattro instrument (MicroMass, Waters, UK) fitted with an electrospray ionisation source. The source and desolvation gas temperature were held constant at 150 °C and 350 °C respectively, with flow rates of 950 and 60 litres of nitrogen per hour.

B6 vitamers and PA were detected using multiple reaction monitoring mode (MRM) and the settings optimised by infusion of each vitamer in proportions of mobile phase according to time of elution off the HPLC column. The mass spectrometer was operated in the positive ion mode for analysis of all B6 vitamers and PA. A single scan segment of 25 minutes provided adequate sensitivity for detection and measurement of all species, giving a total sample run time of 25 minutes. The final chosen parameters for the parent and most abundant daughter ion are as detailed in Table 2. Parent-daughter ion pairs suggested a loss of H2O (18) for PL; loss of ammonia (17) and H2O (18) for PM; loss of 2.H2O (36) for PN; loss of phosphoric acid (98) for PLP; loss of phosphoric acid (98) and ammonia (17) for PMP and loss of 2.H2O (36) for PA. As PNP was not available as a calibration vitamer, an additional transition was added to the MS acquisition file for this compound based on a theoretical fragmentation pattern due to loss of phosphoric acid and water (based on the fragmentation of PLP and PN) (116).

All compounds could be differentiated on the basis of both m/z ratio and retention time. PLP, PMP and PNP had very similar retention times and parent ions that differed by only 1 Da. There was however no cross talk between ion pairs originating from these different analytes. A 25 μL volume of deproteinised plasma was injected every 25 minutes; the column effluent was then delivered to the mass spectrometer with the first 1 minute and last 5 minutes being diverted to waste. MassLynx software was used for data acquisition and analysis.

QUANTIFICATION OF B6 VITAMERS AND PYRIDOXIC ACID

B6 vitamers and pyridoxic acid were quantified in plasma by the addition of a known concentration (100nM) of deuterated internal standard. The amount of PLP and PMP present was calculated as a ratio of the signal area for the vitamer to the signal area for D2 PLP. Similarly D3 PL was used to calculate the amount of PL and PM present, D2 PN for PN and D2 PA for PA.

RESULTS

CALIBRATION CURVES AND LINEARITY

To estimate the linear range of the method, calibration curves in the range 1 – 500 nmol/L were constructed for each vitamer in water and plasma. This range was chosen because it covers the physiological range expected to be encountered in the analysis of human plasma samples. r2 values of equal to or greater than 0.98 were achieved for each vitamer by linear regression (Table 3). Examination of the calibration curves indicated no deviation from linearity at the lowest concentration tested (1 nmol/L) and, at this concentration, the signal to noise ratio was satisfactory for each vitamer (signal-to noise ratio 5:1 or above) making the lower limit of detection for each vitamer in water equal to or less than 1 nmol/L.

PRECISION STUDIES; INTRA- AND INTER-BATCH COEFFICIENT OF VARIANCE AND RECOVERY STUDIES

Assay precision was assessed by calculation of the intrabatch and interbatch coefficient of variance (CV) [CV% = (standard deviation/mean) x 100] in plasma for each vitamer and by recovery of each analyte [recovery % = [(measured concentration-initial concentration)/concentration added] x 100]

Plasma was spiked with medium (50 nmol/L) and high (100 nmol/L) concentrations of each vitamer. One batch was left ‘unspiked’ with endogenous concentrations of B6 vitamers.

10 samples of each concentration (50 nmol/L and 100 nmol/L) were analysed for calculation of the intrabatch CV. The CV for each vitamer was <10% (Table 4).

Interbatch CV’s were calculated in plasma using three samples of each different concentration (50 nmol/L and 100 nmol/L) on five different days over a period of 4 weeks. CV’s for PLP, PMP, PA, PN and PL were <10% but the CV for PM was higher at 17.0% (Table 4). This was felt to be acceptable for use in clinical samples where PM is not usually expected to be present.

Recovery of B6 vitamers from plasma ranged from 68-124% (Table 4). For the three compounds always present and for which accurate measurement is most important (PLP, PL and PA) recoveries are 88 – 116%.

INDICATORS OF ACCURACY

Pyridoxal phosphate of a known concentration in plasma is available from Chromsystems® for use as a quality control standard in their HPLC B6 analysis kit. Analysis of this standard in duplicate on three occasions using the LC-MS/MS method produced comparable results to those of the Chromsystems method (LC-MS/MS method 70; 72; 98 nmol/L; Chromsystems method 68; 67; 86 nmol/L). In addition 39 plasma samples were run in parallel to directly compare the two methods which showed good agreement; r2 = 0.95, p<0.0001 (Figure 2).

STUDY OF MATRIX EFFECTS

The possible matrix effects of plasma on detection of B6 vitamers was studied by measurement of the peak intensities for each vitamer in plasma spiked with differing concentrations of vitamers (13 concentrations, range 3 – 500 nmol/L). These intensities were compared to peak intensities for the vitamers in water where matrix effect % = [(peak area spiked – peak area endogenous)/peak area in water] x100.

Effects of ion suppression were seen for all vitamers in plasma as follows; PL 33%; PM 69%; PN 71%; PA 79%; PMP 86% and PLP 96%. While these figures (particularly those for PL, PM and PN) are lower than for an ideal method, the recovery experiments indicated that the internal standards were adequate to compensate for the matrix effects.

STABILTY OF STANDARDS AND PLASMA SAMPLES

Vitamin B6 is light and temperature sensitive (Saidi and Warthesen 1983). The aqueous B6 standards and spiked pooled plasma samples were handled as detailed above and stored in aliquots at -80°C. Both were found to be stable over a 6 month period.

REFERENCES

Midttun O, Hustad S, Solheim E, Schneede J, Ueland PM. (2005) Multianalyte quantification of vitamin B6 and B2 species in the nanomolar range in human plasma by liquid chromatography-tandem mass spectrometry Clin Chem. Jul;51(7):1206-16

Saidi B and Warthesen J (1983). Influence of pH and light on the kinetics of vitamin B6 degradation. J Agric Food Chem:31:876-80

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