D-768
Methylsulfonylmethane Determination by GC using FLame Ionization Detection
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1.0 Purpose The purpose of this procedure is to define the method for the quantitation and/or identification of Methylsulfonylmethane in raw material and finished product dietary supplements by GC using flame ionization mode. 2.0 Scope This procedure applies to the quantification and identification of MSM in raw materials and finished products. MSM readily detectable by FID on the GC using the direct inject mode. 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 implement this procedure and 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 FID — Flame Ionization Detection 4.2 DGME — Di(ethylene glycol) methyl ether 4.3. MSM —-Methylsulfonylmethane or dimethyl sulfone 4.4 MeOH — Methanol [SOP Standard Operating Procedure SOP No | Rev Page Methylsulfonylmethane Determination by GC using D-768 2 2 of 9 Flame Ionization Detection 5.0 References 5.1 USP 41 Methylsulfonylmethane Monograph 5.2. MV-LAB-18-069, Protocol, Methylsufonylmethane Determination by GC using Flame Ionization Mode 5.3. D-793, SOP, Cryogenic Grinding of Chewable Gels 6.0 Supplies 6.1 Chemicals: All reagents are GC grade or better. 6.1.1 MeOH 6.1.2 DGME 6.1.3 MSM reference standard 6.2 Glassware 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa 6.2.2 Scintillation vials 6.2.3 Mobile phase containers 6.2.4 Volumetric glassware as required by standard and sample preparations 6.3. Disposables 6.3.1 Adjustable pipette tips 6.3.2 Micro-centrifuge tubes 6.3.3. 16mL test tubes [SOP Standard Operating Procedure SOP No | Rev Page Methylsulfonylmethane Determination by GC using | 9-768 ze 3 of 9 Flame Ionization Detection 6.3.4 Disposable plastic luer lock syringe — 3mL, 6mL, or 10mL 6.3.5 Nylon syringe filters, 0.45 um 6.3.6 Weigh paper 6.4 Equipment 6.4.1 Agilent 7890 GC with headspace 6.4.2 Analytical balance 6.4.3 Ultrasonic bath 6.4.4 Vortex 6.4.5 Stir plate 6.4.6 Eppendorf centrifuge 6.4.7 Adjustable pipettes 7.0 Preparation of Diluent, Samples, and Standards 7.1. Diluent 7.1.1 Seale the Diluent preparation as required. 7.1.2 Transfer 0.60 mL of DGME to a 1-L media bottle. 7.1.3 Add 1000 mL methanol, and mix well. Le Standard Preparation 7.2.1 Accurately weigh and transfer about 30 mg of MSM reference standard into a 100-mL volumetric flask. [SOP Standard Operating Procedure SOP No | Riv Page Methylsulfonylmethane Determination by GC using 9768 4 of 9 Flame Ionization Detection 7.2.2 Dissolve in and dilute to volume with Dilue nt 7.3 Sample Preparation 7.3.1 Specific sample testing details are provide] in each produ:ts profile. If a specific testing details section is not aviilable, then fol ov preparation procedure as described below, maintaining 20 iceritration withir tle linear range listed below 7.3.2 The linear range for MSM is 0.1 mg/mL —(.5 mg/ml. 7.3.3 For raw materials: weigh no less than 20 m3 inte a suitably iz:d vcbametric flask of no less than 25 mL volume to gen »ré te an analyte con:e1 tratior. that is within the validated linearity range. Dilute .o volurie with Dilu sn, and sonicate for 10 min. 7.3.4 For solid and liquid dose finished producti: “ombine and hon io; enize no less than ten dosage units. Based on the labi:l claim and fill wiigat (capsules), serving size (powders) or tablet weigkt pe’ case, ‘weigh no less 11an °C mg of the pooled dosages into a suitably sized voluicetric flask of no le: s than 25 mL to generate an analyte concentration that s withia the velida ed linewr range. Dilute to volume with Diluent, and sonicate fer 10 x0in. 7.3.5 For chewable gels (gummies), homogenize at least 10 dosage u tits according to the procedure outlined in D-793 Cryogenic Grinding of Chewat le Gels. Quickly weigh a portion of the pooled and homogenized dosages int». beaser. Use several small portions of Diluent to completely transfer the sampl: into a suitably sized volumetric flask to generate an analyte cone ntration that is within the validated linear range. Dilute to volume, and son:cate for 10 min. 7.3.6 To manage large volumes, the sample can be initially dissolec in a smaller volume and a portion further diluted using Diluent to br nj; the analyze [SOP Standard Operating Procedure SOP No Rev Page D-768 Methylsulfonylmethane Determination by GC using 5 of 9 Flame Ionization Detection concentration into the linear range of measurement. Equilibrate the standard solution to room temperature prior to performing further dilution. 7.3.7 If particulates remain in the final sample preparation, a portion may be centrifuged at 10,000 rpm for 5 min prior to HPLC analysis. Alternatively, the sample may be filtered through a 0.45 um membrane discarding the first 3 — 4 mL. 8.0 Test Conditions 8.1 Column — Phenomonex ZB-1, 0.53mm x 30m, 5um, GC column, or equivalent. 8.2 Carrier gas— Helium 8.3 Inlet Liner — Restek, 4.0mm ID x 6.3mm OD x 78.5mm length straight liner with wool, or equivalent. 8.4 Flow Rate — 5.0mL/min 8.5 Temperature 8.5.1 Injector — 250°C 8.5.2 Column — 120°C 8.5.3 Detector — 250°C 8.6 Run Time 8.6.1 Blanks and Standards - 7 min 8.6.2 Samples - 30 min (longer may be required if samples exhibit peaks at long time) 8.7 — Spit ratio - 2:1 8.8 Injection volume — 1uL [SOP Standard Operating Procedure SOP No Rev Page Methylsulfonylmethane Determination by GC using D-768 6 of 9 Flame Ionization Detection 8.9 Septum purge — 3 mL/min 8.10 Air flow — 400 mL/min 8.11 Hydrogen flow — 40 mL/min 8.12 Makeup flow — 30 mL/min (column + makeup = constant) 8.13 Example Sequence 8.13.1 Perform at least one injection of a Blank (Diluent). 8.13.2 Perform five injections of the Working Standard. 8.13.3 Perform a single injection of each Sample Preparation. 8.13.4 Perform a single injection of the Working Standard after every six samples and at the end of the run. 8.14 Retention Times 8.14.1 MSM — about 4.8 min 8.14.2 DGME - about 6.0 min 8.15 System Suitability 8.15.1 The %RSD of the peak area ratio for five injections of the Working Standard is NMT 2.0%. 8.15.2 The %RSD of the peak area ratio for all injections of the Working Standard is NMT 2%. [SOP Standard Operating Procedure SOP No | Rey Methylsulfonylmethane Determination by GC using D-768 2 ; 5 Flame Ionization Detection 9.0 Example Calculation R, . Wiss XP . Vax... FW % Label (FP) or % Assay (RM) = — xX —— ce x 100 Rs Viste Sply, LA Ry Sample peak area ratio R, Mean Working Standard peak area ratio (5 injections) Wterq Weight of reference standard used to prepare Working Standard (mg) PF Purity of reference standard from the CoA (decimal) Veiqg Volume of Working Standard (mL) Vsp, Volume of Sample Solution including dilutions (mL) Spl,,, Sample weight (mg) FW Theoretical fill/tablet weight (mg, use 1 for raw materials) LA Label amount (mg, use 1 for raw materials) 10.0 Example Chromatograms 10.1 Blank Blank : aD. OsWw 200 a Q 1507 < 100: 0G N» B«LeOeW 8N6 nL 8 ated 6 of 4 #46 +2 25 #8. 968 4. 461 °8 “S85. 6 65° °7 Time [min] [SOP Standard Operating Procedure SOP No | Rev e Methylsulfonylmethane Determination by GC using | -768 ze 5 re Flame Ionization Detection 10.2 Working Standard WSTD 19AS107 : : 200: % G} ; vfoe)2 < 400; 505 a QE ® ” Oo +O i) 0 0.5 1 1.5 2 2.5 3 3.5 4 45 5 5.5 6 7 Time [min] 10.3. Sample Preparation 820397 T=24m A N jan} 200; iNx 2c= HWes D 1507 rs 1005 KF OOOO"O |p ie] i 0 ‘ mn iil iid ¥ + qT T LU T qT 7 U qT T LU q qT ' y T T T Li Ul 123 45 67 8 9 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Time [min] [SOP Standard Operating Procedure SOPNo | Rev f Methylsulfonylmethane Determination by GC using D-768 2 9 a Flame Ionization Detection 11.0 Revision History | Rev | Date | Description of Changes | CCR # | By | |-----|----------|------------------------|-------|----| | 0 | 01/02/19 | New N/A J. Maignan Added reference to validation, include linear range, simplify diluent and standard prep. Added short run time for standards and | - | - | | 1 | 09/07/22 | blanks, add retention times and example chromatograms, update CC- system suitability and calculations. Removed “requirements” section. Removed unnecessary information and align with current SOP format. Added instruction to refer to test details for product | 22-0365 | S. Sassman | | 2 | 05/17/23 | specific sample preparation. Added specific sample preparation CC- instruction for different dosage forms, Changed system suitability criteria from NLT 5% to NLT 2%. Changed logo. | 23-0230 | S. Sassman |