Analysis of Thiamine PyroPhosphate and Pyridoxal-5’-Phosphate in whole blood using the UHPLCMS-8050 and the MagnaMedics MagSi-VitB1/B6PREP kit. Dennis JA vd Heuvel1, Erik JB Ruijters2, Bart Jansen1 1Shimadzu Benelux B.V., NL 2MagnaMedics Diagnostics B.V., NL

1. Introduction 3. Results and discussion Vitamin B1, thiamin, plays an important role in the metabolic pathway in the human body. The biological active form is Thiamine Pyrophosphate (TPP). The water soluble vitamin acts as a coenzyme for the enzymatic degradation of glucose in the citric acid cycle. A non-varied diet or 3-1. Method results malnutrition can quickly lead to a deficiency which can result in inter alia depression, muscle Both compounds showed excellent linearity (r2> 0.999) in a clinically relevant concentration range weakness and tachycardia. (TPP: 11.8 – 1176 nmol/L; PLP 20.2 – 2023 nmol/L). The LOD and LOQ were 2.7 nmol/L and 8.2 Vitamin B6 has multiple forms. The biological active form in the human cell is Pyridoxal-5’- phosphate (PLP). The water soluble vitamin acts as a coenzyme in the formation of aminoacids, nmol/L for TPP, respectively and 0.6 nmol/L and 1.8 nmol/L for PLP. amines and peptides. In case of a deficiency the other B vitamins will also be deficient. A PLP Compound Concentration SD %RSD deficiency can occur due to chemotherapy, alcoholism, pregnancy and kidney failure. To this day [nmol/L] these two vitamins (TPP and PLP) are predominantly analysed with HPLC and fluorescence detection. These methods are performed with excessive sample preparation including pre- or TDP 11.8 0.74 6.2 postcolumn derivatisation using toxic reagents and have relatively long runtimes. Due to the 276 15 5.6 rising numbers of patient samples in clinical laboratories there is need for a simple and fast 1176 32 2.6 chromatographic method without excessive sample preparation. The aim of this study was therefore to set up a simple and fast UHPLC method with mass spectrometric detection, with PLP 20.2 1.4 6.8 minimal sample preparation, resulting in a total solution. 443 25 5.6 2023 113 5.7

Table 1 – Coefficient of variation for TDP and PLP at three levels

Y=0.0157027x+0.0401776 Y=0.00577536x+0.0154340 R2=0.9993500 R2=0.9991187

Thiamine PyroPhosphate Pyridoxal-5’-Phosphate 2. Method 2-1. Sample preparation a b From whole blood samples, calibrator or control samples only 25µl of sample was transferred to a 96-well microtiter plate which was positioned above a magnet. 10 µL of internal standard mix Figure 1a,b – Calibration curve TPP (11.8 – 1176 nmol/L) (a); Calibration curve PLP (20.2 - 2023 PREP (containing d3-TPP and d3-PLP) and 40 µL MagSi-VitB1/B6 bead mix was added to the nmol/L) (b) sample and mixed. The proteins were precipitated by the addition of 200 µL precipitation solution followed by intense aspiration and dispensing of the mixture. After magnetic separation 90 µL of the supernatant was transferred to a HPLC vial and mixed with acetonitril.

2-2. LC-MS/MS analysis 10 µL of the supernatant was analysed on a Nexera X2 binary UHPLC system (Shimadzu, Japan) and coupled to a tandem quadrupole mass spectrometer (LCMS-8050, Shimadzu, Japan). TPP was measured with MRM transition 424.9>122.2 and PLP with MRM transition 247.9>150.0. For both compounds two reference ions were measured simultaneously and the ratio between the main transition and the reference transitions was established and set in the method. The deuterated internal standards were measured with MRM transitions 427.9>125.2 a b c d

(d3-TPP) and 250.9>153.0 (d3-PLP). Figure 2 a,b,c – Chromatogram of lowest calibration point TPP (a), lowest calibration point PLP UHPLC METHOD (b) and a patient sample (c: TPP 182 nmol/L; d: PLP 127 nmol/L) Column Phenomenex F5, 100 x 3 mm, 2.6 µm Column temperature [°C] 15

Mobile phase A: Acidified H2O

B: CH3CN Injection volume [µL] 10 Flow rate [mL/min] 0.3 MS/MS METHOD

Nebulizer gas [L/min] 2 (N2) Heating gas [L/min] 10 (Air) Figure 3 – Nexera X2 binary UHPLC system coupled to the LCMS 8050 tandem quadrupole

Drying gas [L/min] 5 (N2) mass spectrometer. Interface temperature [°C] 300 Desolvation line [°C] 250 Heat b;ock temperature[°C] 400 4. Conclusion Interface voltage [kV] 4 • The developed method with simple and fast sample preparation is an appropriate method for Dwell time [ms] 30 Pause time [ms] 3 detection of TPP and PLP in whole blood samples. Ionization ESI, positive • Both compounds, TPP and PLP showed excellent linearity (r2> 0.999) in a clinically relevant Scan Type MRM range. Compound Target (T) / Internal MRM MRM MRM Retention time • Full sample preparation for a 96 well plate takes 30 minutes, total runtime on the LCMS/MS is Standard (I) Quantifier Qualifier 1 Qualifier 2 [min] 3 minutes per sample. • Extensive method validation and comparison is under progress. TDP T 424.9>122.2 424.9>304.0 424.9>81.1 1.60 • Due to the fast and automated sample pretreatment and short analysis time this total solution is PLP T 247.9>150.0 247.9>94.1 247.9>122.05 1.79 applicable for high numbers of patient samples offered at clinical laboratories. • The cost per sample is relatively low in comparison to accepted standard methods, due to the d3-TDP I 427.9>125.2 - - 1.63 limited sample pretreatment without centrifugation, short run times and no need for expensive d3-PLP I 250.9>153.0 - - 1.82 reagents.