D-1022
Determination of Water Soluble Vitamins by LC-MS
Original Document
Scanned document (image-only PDF)
Extracted Text
Searchable text extracted from PDF
1.0 Purpose The purpose of this procedure is to define the method for the determination of water soluble vitamins in raw materials and finished products by LC-MS. 2.0 Scope This procedure applies to the determination of vitamin B1 (thiamin hydrochloride, thiamin mononitrate), vitamin B2 (riboflavin), vitamin B3 (niacinamide, nicotinic acid), vitamin B5 (pantothenic acid, calcium pantothenate), vitamin B6 (pyridoxine, pyridoxine hydrochloride), vitamin B7 (biotin), and vitamin B9 (folic acid) in the QC laboratory at Ion Labs. 3.0 Responsibility 3.1 It is the responsibility of QC Chemists to follow this procedure. 3.2 ‘It is the responsibility of QC Laboratory Management to ensure that this procedure is being followed. 3.3. ‘It is the responsibility of QC Laboratory Management and/or Analytical Development to keep this procedure aligned with current practices. 4.0 Definitions 4.1 LC-MS -— Liquid chromatography — mass spectrometry 4.2 QC — Quality control 4.3 ACN — Acetonitrile [SOP D-102 | Page 2 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 I Hate SEIS 44 DMSO - Dimethylsulfoxide 4.5 Na2x2EDTA-2H20 — Ethylenediaminetetraacetic acid disodium salt dihydrate 4.6 HO - Deionized water (>18MQ-cm) 5.0 References 5.1 PRTCL-23-0088, Protocol, Validation of a Method for the Determination of Water Soluble Vitamins by LC-MS 5.2 PRTCL-24-0031, Protocol, Validation of a Method for the Determination of Water Soluble Vitamins by LC-MS 5.3 D-903, SOP, Conversion Factors Used in Analytical Determinations and New Product Formulations 6.0 Supplies 6.1 Chemicals 6.1.1 Reference standards (traceable to USP or other pharmacopeia preferred) 6.1.1.1 Thiamin hydrochloride 6.1.1.2 Pyridoxine hydrochloride 6.1.1.3 Niacinamide 6.1.1.4 Nicotinic acid 6.1.1.5 Calcium-D-pantothenate 6.1.1.6 Folic acid 6.1.1.7 Biotin [SOP D-102 | Page 3 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 : Bases OFtS 6.1.1.8 Riboflavin 6.12 Isotopically labelled internal standards (100y.2/mL solution or neat solid) 6.1.2.1 Thiamin-?C3 6.1.2.2 Pyridoxine-'°C3 6.1.2.3 Niacinamide-!?C¢ 6.1.2.4 Nicotinic acid-!C¢ 6.1.2.5 Pantothenic acid-'°C3,5N 6.1.2.6 Folic acid-'Cs,N 6.1.2.7 Biotin-D4 6.1.2.8 Riboflavin-!3C4,!5N> ACN (HPLC or LC-MS grade as specified in Section 7) Ammonium formate (HPLC or LC-MS grade as specified in Section 7) Ammonium acetate (HPLC grade) Citric acid (ACS reagent grade) Na2EDTA-2H20 (ACS reagent grade) 6.1.8 Formic acid (LC-MS grade) 6.2 Glassware and Disposables 6.2.1 Class A volumetric glassware as required for standard and sample preparations 6.2.2 HPLC vials, 2mL with screw-cap enclosures and septa [SOP D-102 | Page 4 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 | 1 Page dot 1s 6.2.3 Tips for adjustable pipettes 6.2.4 0.22 or 0.45 um nylon or PVDF syringe filters 6.2.5 10-mL plastic syringes with luer lock fitting 6.3 Equipment 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, and column oven 6.3.2 Agilent Ultivo mass spectrometer using MassHunter software 6.3.3 Analytical balance 6.3.4 Micro balance 6.3.5 Sonicator 6.3.6 Wrist action shaker 6.3:7 Adjustable pipettes 7.0 Preparation of Mobile Phase, Extraction Solvent, Standards, Internal Standards, and Samples 7.1 Mobile Phase A (20 mM ammonium formate + 0.05 % formic acid in H20) 7.1.1 Transfer 1000 mL of H20 to a suitable container. 7.1.2 Add 1.26 g of ammonium formate (LC-MS grade). V1.3 Add a stir bar, and adjust to pH 3.80 + 0.05 using formic acid. 7.2. Mobile Phase B (ACN) 7.2.1 Use 100% ACN (LC-MS grade). [SOP D-102 | Page 5 of 15] Standard Operating Procedure SOP No| Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 . Hazeoionts 7.3. Extraction Solvent (70% buffer containing 100 mM ammonium acetate, 25 mM citric acid, and 5 mM Na2EDTA-2H20 / 30% ACN) Note: The Extraction Solvent for extraction of nicotinic acid from samples is 100% DMSO. For all other analytes and for preparation of standard and internal standard nicotinic acid solutions, prepare as outlined below. Tdel Transfer 5.4 g of ammonium acetate to a 1000-mL bottle. 7.3.2 Add 3.36 g of citric acid. 7.3.3. Add 1.3 g of NaEDTA-2H20. 7.3.4 Add 700 mL H20. 1.35 Add 300 mL ACN (HPLC grade). 7.3.6 Stir until completely dissolved, and equilibrate to room temperature. 7.4 Diluent (20 mM ammonium formate + 0.005% formic acid) 7.4.1 Transfer 1000 mL of H20 to a suitable container. 7.4.2 Add 1.26 g of ammonium formate (HPLC grade). 7.4.3. Add 50 uL of formic acid. 7.4.4 Mix until completely dissolved. 7.5 Internal Standard Stock Solution (2.5 g/mL) 7.5.1 The Internal Standard Stock Solution is prepared from individual 100 pg/mL isotopically labelled standards which are commercially available. Alternatively, 100 g/mL solutions may be prepared from solid materials by dissolving 1 mg of isotopically labelled standard in 10 mL of Extraction Solvent. [SOP D-102 | Page 6 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 | 1 | Page6 of 15 7.5.2 Transfer 1.0 mL of each 100 g/mL isotopically labelled internal standard solution into a 50-mL low-actinic (red) volumetric flask. 7.5.3 Add 34 mL of Extraction Solvent, and mix well. 7.5.4 Purge with argon, and store at -20°C. The solution has an expiry of one year. 7.6 Internal Standard Working Solution (100 ng/mL) 7.6.1 Transfer 1.0 mL of Internal Standard Stock Solution to a 25-mL low-actinic (red) volumetric flask. 7.6.2 Dilute to volume using Diluent. 7.6.3 Store at 4°C. The solution has an expiry of one month. 7.7. Stock Standard (~10 mcg/mL) dided Accurately weigh and transfer about 5 mg of each reference standard into a 500- mL low-actinic (red) volumetric flask. 7.7.2 Dilute to volume using Extraction Solvent. 7.7.3. Adda stir bar, and stir for 1 hour or until completely dissolved. 7.7.4 Store at 4°C. The solution has an expiry of 10 days. 7.8 Working Standard (~0.3 mcg/mL) 7.8.1 The Working Standard is prepared fresh on the day of use. 7.8.2 Remove the Stock Standard from refrigeration, and stir for at least one hour. 7.8.3 Transfer 3.0 mL of Stock Standard to a 100-mL low-actinic (red) volumetric flask. [SOP D-102 | Page 7 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 4 pagewiob ls 7.8.4 Dilute to volume with Diluent. 7.8.5 Combine 0.5 mL of the Working Standard with 0.5 mL of the Working Internal Standard in an HPLC vial, and vortex to mix. 7.9 Stock Sample Preparation 794 Specific sample testing details are provided in each products profile. If a specific testing details section is not available, then follow preparation procedure as described below, maintaining concentration within the linear range of this method. 7.9.2 | The Stock Sample is prepared fresh on the day of use. 7.9.3 The default sample weight is 1.0 g. 7.9.3.1 Finished products with large dosage units and/or very small amount of target analyte may require larger sample size. 7.9.3.2 For raw materials or finished products with a very large amount of target analyte, the sample size may be decreased with a minimum recommended weight of 50 mg. 7.9.4 Based on the label claim or potency, ensure that the analyte concentration in the Stock Sample does not exceed the solubility listed below: 7.9.4.1 Vitamin B1 (thiamin) 5 mg/mL 7.9.4.2 Vitamin B2 (riboflavin) 0.4 mg/mL 7.9.4.3 Vitamin B3 (niacinamide) 5 mg/mL 7.9.4.4 Vitamin B3 (nicotinic acid) 5 mg/mL 7.9.4.5 Vitamin B5 (pantothenic acid) 5 mg/mL [SOP D-102 | Page 8 of 15] Standard Operating Procedure SOP No| Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 I Fagedobts 7.9.4.6 Vitamin B6 (pyridoxine) 5 mg/mL 7.9.4.7 Vitamin B7 (biotin) 0.5 mg/mL 7.9.4.8 Vitamin B9 (folate) 0.1 mg/mL 7.9.5 Ensure that the sample is homogeneous prior to weighing. 7.9.5.1 For capsules, combine the fill material from at least 10 dosage units and homogenize in a mortar and pestle if necessary. 7.9.5.2 For tablets, combine at least 10 dosage units and homogenize in a mortar and pestle. 7.9.5.3 For chewable gels (gummies), homogenize at least 10 dosage units as outlined in D-793. 7.9.6 Transfer 1.0 g of sample to a 100-mL low-actinic (red) volumetric flask. 7.9.7 Add about 65 mL of Extraction Solvent (use 100% DMSO as extraction solvent for nicotinic acid). 7.9.8 Shake on a wrist-action shaker for 30 min. 7.9.9 Dilute to volume with Extraction Solvent (or DMSO for nicotinic acid), and shake vigorously. 7.9.10 Ifthe sample is laden with particulate matter, filtration through a 0.22 or 0.45 um membrane (discarding 2 — 3 mL before collecting the sample for further dilution) may be necessary prior to further dilution of the sample. 7.10 Working Sample Preparation 7.10.1 The linear range of the method for each analyte is listed below. The working sample preparation must be within the linear range of the method. [SOP D-102 | Page 9 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 1 Hage S08 16 7.10.1.1 Vitamin B1 (thiamin) 14-700 ng/mL 7.10.1.2 Vitamin B2 (riboflavin) 15 — 773 ng/mL 7.10.1.3 Vitamin B3 (niacinamide) 82 — 824 ng/mL 7.10.1.4 Vitamin B3 (nicotinic acid) 13 — 1600 ng/mL 7.10.1.5 Vitamin BS (pantothenic acid) 16-793 ng/mL 7.10.1.6 Vitamin B6 (pyridoxine) 31 —781 ng/mL 7.10.1.7 Vitamin B7 (biotin) 13 — 1600 ng/mL 7.10.1.8 Vitamin B9 (folate) 2 — 1228 ng/mL 7.10.2 The Working Sample is prepared fresh on the day of use. 7.10.3 Dilute the Stock Sample to the target concentration using Diluent. Multiple dilutions may be required. 7.10.4 Filter through a 0.22 or 0.45 um membrane (discarding 2 — 3 mL before collecting the sample for analysis). If the stock sample was filtered, filtration of the working sample is not required. 7.10.5 Combine 0.5 mL of the Working Sample with 0.5 mL of the Working Internal Standard in an HPLC vial, and vortex to mix. 7.11 Instrument Method Parameters 7.11.1 Column: InfinityLab Poroshell 120 EC-C18, 2.7 um, 2.1 mm x 150 mm 7.11.2 Sampler 7.11.2.1 Injection Volume: 5 wL 7.11.2.2 Enable Needle Wash: Selected [SOP D-102 | Page 10 of 15] Standard Operating Procedure SOP No Rev Determination of Water Soluble Vitamins by LC-MS D-1022 Page 10 of 15 7.11.2.3 Mode: Flush Port 7.11.2.4 Time: 10 sec 7.11.2.5 Sample Flush-Out Factor: 5.0 times injection volume 7.11.2.6 Overlapped Injection Mode: Off 7.11.3 Binary Pump 7.11.3.1 Flow Rate: 0.2 mL/min 7.11.3.2 Gradient Time (min) %A %B 0.0 95 5 9.0 60 40 9.1 95 5 12.0 95 5 7.11.4 Column Oven 7.11.4.1 Temperature: 35 °C 7.11.5 QQQ 7.11.5.1 Acquisition TALSL1 Scan type: (MRM TLS Cycle Time: 600 ms Taddvd1s3 Polarity: Positive 711.5) 14 Acquisition Parameters for Target Analytes Note: Retention times and/or window may be adjusted if peaks shift outside of the listed retention time window. [SOP D-102 | Page 11 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 - Page diols MS1 |Precursor} MS2 | Product} RT |Window) Frag CE Anaal e Iatsid | Res «| (evz) | Res. | -Gn/zy |tmin). | @nin). | (vy thiamin | thiamin-'°C3| Unit | 265 Unit 122: | 205 1.2 76 13 pyridoxine ina Unit | 170 Unit 134) (396 |° 1.0 86 pa niacinamide nae Unit | 123 Unit go | 260 | 1.0 106 | 21 nicotinic acid Ter ta Unit | 124 Unit BO | oO iiss 75 20 pantothenic | pantothenic . asia acid-3C; Unit 220 Unit 90 3.65 1.9 96 11 folic acid teae Unit | 442 Unit | 295 | 660 | 2.0 95 15 biotin biotin-D4 Unit 245 Unit 227 9.10 2.0 70 10 riboflavin eee Unit | 377 Unit | 243 | 850 | 18 129 | 22 4, 2 7.11.5.1.5 Acquisition Parameters for Internal Standards Int Std MS1 | Precursor} MS2 | Product | RT |Window| Frag CE Res (m/z) Res (m/z) | (min) | (min) (V) (V) thiamin-°C3 Unit | 268 Unit 122 | 2.05 12 76 13 pyridoxine-?C3 Unit 173 Unit 137 2.20 1.0 86 ad niacinamide-!7Ce¢ Unit 129 Unit 85 2.60 1.0 106 21 nicotinic acid-'?C¢ Unit 130 Unit 85 2.30 LS 75 20 pantothenic acid-!3C3 Unit | 224 Unit 94 3.65 1.9 96 11 folic acid-!Cs,!5N Unit 448 Unit 295 6.60 2.0 95 15 biotin-D4 Unit | 249 Unit 231 9.10 2.0 70 10 riboflavin-!3C4,!°N2 Unit | 383 Unit 249 | 8.50 1.8 129 22 [SOP D-102 | Page 12 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 | 1 | Page 12 of IS 7.11.5.1.6 Source Parameters Nebulizer Sheath Capillary Nozzle Source Gas oeT o: n panty Pressure BearC) Flow Voltage Voltage (psi) P (L/min) (V) (V) AJS ESI 350 7 2 400 12 2500 0 7.12 Recommended Sequence 7.12.1 Make at least 2 injections of Diluent. 7.12.2 Make 5 injections of the Working Standard. 7.12.3 Make a single injection of each Working Sample. 7.12.4 Make a single injection of the Working Standard after every six samples and at the end of the run. 7.13 System Suitability Requirements 7.13.1 The %RSD for five consecutive injections of Working Standard is NMT 2.0%. 7.13.2 The %RSD for all injections of Working Standard is NMT 3.0%. 7.13.3 If significant interference is present in the diluent injection (the relative response is greater than 0.5% of that in the sample injection), it must be subtracted from the sample result. 7.14 Column Cleaning 7.14.1 It is recommended to clean the column after every run. 7.14.2 Clean the column at 0.2 mL/min with Mobile Phase A / Mobile Phase B (20/80) for at least 30 min. 7.15 Column Storage 7.15.1 Store the column in Mobile Phase A / Mobile Phase B (20/80). [SOP D-102 | Page 13 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 i Rage ts of 15 8.0 Example Calculation % assay = = x a a = x a x MULT x 100 Ry Sample relative response Rg Mean standard relative response Wtsta Weight of reference standard in mg Vota Volume of the standard preparation accounting for dilutions in mL P Purity of the reference standard in decimal format 55 Serving size: Weight of a single dosage unit in mg or 1 for raw materials. Splwe Sample weight in mg Vspi Volume of the sample preparation accounting for dilutions in mL LA Label amount in mg per dose or 1 for raw materials MULT Multiplier (from D-903) [SOP D-102 | Page 14 of 15] Standard Operating Procedure SOP No | Rev Determination of Water Soluble Vitamins by LC-MS | D-1022 | 1 | Page 14 of IS 9.0 Example Chromatograms + MRM (265.0 -> 122.0) thiamin WSTD + MRM (268.0 -> 122.0) thiamin-13C3 WSTD 2 x105+ 4326200. 2 x105] 10. 3 4- 3 i S os Co 24 0 0 T T T T T T T T T T T 1.6 1.8 2 22 24 2.6 1.6 1.8 2 2.2 24 Acquisition Time (min) Acquisition Time (min) + MRM (170.0 -> 134.0) pyridoxine WSTD + MRM (173.0 -> 137.0) pyridoxine-1W3SCT3D 2 x105 5266700. 4 x1054 1294200. b— J J ‘ad 8 14 0 0 T T T T T T T T T T 18 2 2.2 24 26 18 2 2.2 24 26 Acquisition Time (min) Acquisition Time (min) + MRM (123.0 -> 80.0) niacinamide WSTD + MRM (129.0 -> 85.0) niacinamide-13C6 WSTD 2 x105- 3058400. 2 x105, 857820. p=] — 6 24 S 054 0 0 T T T T T T T T T T 2.2 24 26 2.8 3 22 24 26 28 3 Acquisition Time (min) Acquisition Time (min) + MRM (124.0 -> 80.0) nicotinic acid WSTD + MRM (124.0 -> 80.0) nicotinic acid WSTD 2 x104 387300. 2 x104 387300. 3 7 3 [ (ae) 4 fae) Pe 2- 24 i) 0 16 #18 #2 22 24 26 28 1 te 2 22 24 26 28 Acquisition Time (min) Acquisition Time (min) + MRM (220.0 -> $0.0) pantothenic acid W/STD + MRM (224.0 -> 94.0) pantothenic acid-13C3,15N WSTD 2 x104- 216230. 2 x1034 70348. = 3 4 é 1 a % 0 0 a a hae Sa ae ae 7 ‘SI I AE, a | T 28 3 32 34 36 38 4 42 44 28 3 32 34 36 38 4 42 44 Acquisition Time (min) Acquisition Time (min) + MRM (442.0 -> 295.0) folic acid WSTD + MRM (448.0 -> 295.0) folic acid-13C5, 15N WSTD 2 x103| 8122.0 2 «1027 2564.0 Fe} 3 24 oO 05-4 oO : T T T . T T T T T 6 6.5 7 6 6.5 7 73 Acquisition Time (min) Acquisition Time (min) [SOP D-102 | Page 15 of 15] Standard Operating Procedure SOP No| Rev Determination of Water Soluble Vitamins by LC-MS _ | D-1022 1 Page ts ob1S + MRM (245.0 -> 227.0) biotin WSTD + MRM (245.0 -> 227.0) biotin WSTD 2 «1057 650230. 2 x1057 650230. Bo 4 ee CO 0.754 C 0754 0.5- 0.54 0.25- 0.25- 0 i) LI q q q T i T T J q qT y qT i q UJ 8 8.2 84 86 88 9 92. 94 8 8.2 84 86 88 ] 92 94 Acquisition Time (min) Acquisition Time (min) + MRM (377.0 -> 243.0) riboflavin WSTD + MRM (383.0 -> 249.0) riboflavin-13C4,15N2 WSTD 2 x1037 44507. 2 x1034 12480. 3 54 . O 25. 4.4 0 0 78 8 82 84 86 88 9 92 78 8 82 84 86 88 9 92 Acquisition Time (min) Acquisition Time (min) 10.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 01/12/24 | New procedure. N/A S. Sassman Add biotin and nicotinic acid as analytes, change mobile phase | - | - | | 1 | 06/06/24 | prep for more careful control of pH, add preference for CC- traceable standards, add grade requirements for chemicals. | 24-0257 | S. Sassman |