B-Hydroxy-B-Methylbutyrate and B-Hydroxybutyrate Determination by HPLC using UV-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation and/or identification
 
 of B-Hydroxy-B-Methyl Butyrate (HMB) and B-Hydroxy Butryrate (BHB) in raw materials and
 
 finished product dietary supplements using HPLC and UV/VIS spectrophotometry.
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of HMB, along with its analogous
 
 forms, and BHB along with its analogous forms in raw materials and finished products. BHB
 and HMB are poor chromophores and were measured at 214 nm. Other wavelengths can be used
 
 if interferences are present. 
 
 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 the QC Laboratory Management and/or Analytical Development
 to keep this procedure aligned with current practices. 
 
 4.0 Definitions 
 
 4.1 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.2. H3POs4-— Phosphoric Acid 
 
 4.3, HCl—Hydrochloric Acid 
 
 4.4 ACN — Acetonitrile 
 
 4.5 | KH2PO4- Monobasic Potassium Phosphate 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 
 p-Hydroxy-B-Methylbutyrate and B- D-760 
 Page 
 Hydroxybutryrate Determination by HPLC using 2 of 9 
 UV/VIS Spectroscopy 
 
 4.6 H20O —Water 
 
 4.7 BHB- -Hydroxy Butryrate; 3-Hydroxybutanoic acid 
 
 4.8 HMB --Hydroxy-B-Methyl Butyrate ; B-hydroxyisovaleric acid 
 
 5.0 References 
 
 521 MV-LAB-18-135, Protocol, f-Hydroxybutyrate and B-Hydroxy-B-Methylbutyrate
 
 Determination using HPLC with UV/VIS Spectroscopy 
 
 5.2. D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 6.1.1 H20 © 18.2 MQ-cm) 
 
 6.1.2 ACN 
 
 6.1.3 H3PO4 
 
 6.1.4 HCl 
 
 6.1.5 KH2PO4 
 
 6.1.6 BHB reference standard 
 
 6.1.7 HMB 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 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 p-Hydroxy-B-Methylbutyrate and B- D-760 2 Page 
 Hydroxybutryrate Determination by HPLC using 3 of 9 
 UV/VIS Spectroscopy 
 
 6.3.1 Pipette tips for adjustable pipettes 
 
 6.3.2 1.5mL microcentrifuge tubes 
 
 6.3.3 16mL test tubes 
 
 6.3.4 Disposable plastic luer lock syringe — 3mL, 6mL, or 10mL 
 
 6.3.5 Nylon syringe filters, 0.45um 
 
 6.3.6 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 
 and UV detector with a chromatographic data handling system 
 
 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 Mobile Phase, Diluent, Samples, and Standards 
 
 7.1 Buffer —0.05M KHpPOs (aq) 
 
 TAsl Transfer 6.803 g of KH2POs, to a 1000-mL mobile phase bottle.
 
 7.1.2 Add 950 mL of H20. 
 
 7.1.3 Adjust to pH 2.9 + 0.5 with H3POu. 
 
 7.1.4 Transfer to a 1000-mL volumetric flask and dilute to volume with H20.
 
 7.2 Mobile Phase A — Buffer and ACN (95:5) 
 
 Toast Transfer 50 mL of ACN to a 1000-mL mobile phase bottle. 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 p-Hydroxy-B-Methylbutyrate and B- D-760 2 Page 
 
 Hydroxybutryrate Determination by HPLC using 4 of 9 
 UV/VIS Spectroscopy 
 
 7.2.2 Add 950 mL of Buffer, and mix well. 
 
 7.3. Mobile Phase B — Acetonitrile and H2O (80:20) 
 
 7.3.1 Transfer 800ml of ACN to a 1000-mL mobile phase bottle. 
 
 7.3.2 Add 200ml of H20, and mix well. 
 
 7.4 Diluent-0.1N HCl 
 
 7.4.1 Transfer 900mL of H20 to a 1000-mL mobile phase bottle. 
 
 7.4.2 Add 100mL of 1N HCl. and mix well. 
 
 7.5 Standard Preparation 
 
 7.5.1 The linear range of the method for each analyte is listed below. All standard and
 
 sample preparations must be within the linear range of the method.
 
 7.5.1.1 HMB -—0.2 to 1.6 mg/mL 
 
 7.5.1.2 BHB-— 0.04 to 1.0 mg/mL 
 
 7.5.2 The standard is prepared by weighing no less than the minimum weight of the
 analytical balance, then bringing up to the final volume in an appropriate
 
 volumetric flask using dissolution buffer and sonicating for 10 minutes.
 
 7.5.3 To manage large volumes, the standard can be initially prepared at a higher
 concentration and further diluted into the linear range using Diluent. Equilibrate
 
 the standard solution to room temperature prior to performing further
 
 dilution. Dilutions can be made using volumetric glassware and/or adjustable
 pipettes. Specific standard concentrations will approximate the concentration
 
 expected to be found in the product being tested based on the sample dilution and
 calculated from the label. Dilutions can be prepared in HPLC vials.
 
 7.6 Sample Preparation 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 
 B-Hydroxy-B-Methylbutyrate and B- D-760 2 Page 
 Hydroxybutryrate Determination by HPLC using 5 of9 
 UV/VIS Spectroscopy 
 
 7.6.1 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 listed above.
 
 7.6.2 For raw materials: weigh no less than 20 mg into a suitably sized volumetric flask
 of no less than 25 mL volume to generate an analyte concentration that is within
 
 the validated linearity range. Dilute to volume with Diluent, and sonicate for 10
 
 min. 
 
 7.6.3 For solid and liquid dose finished products: Combine and homogenize no less
 than ten dosage units. Based on the label claim and weight per dose, weigh no
 
 less than 50 mg of the pooled dosages into a suitably sized volumetric flask of no
 less than 25 mL to generate an analyte concentration that is within the validated
 
 linear range. Dilute to volume with Diluent, and sonicate for 10 min.
 
 7.6.4 For chewable gels (gummies), homogenize at least 10 dosage units according to
 
 the procedure outlined in D-793 Cryogenic Grinding of Chewable Gels. Quickly
 weigh a portion of the pooled and homogenized dosages into a beaker. Use several
 
 small portions of Diluent to completely transfer the sample into a suitably sized
 volumetric flask to generate an analyte concentration that is within the validated
 
 linear range. Dilute to volume, and sonicate for 10 min. 
 
 7.6.5 To manage large volumes, the sample can be initially prepared at a higher
 
 concentration and further diluted into the linear range using Diluent. Equilibrate
 the sample solution to room temperature prior to performing further
 
 dilution. Dilutions can be made using volumetric glassware and/or adjustable
 pipettes. Dilutions can be prepared in HPLC vials. 
 
 7.6.6 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 pm membrane discarding the first 3 - 4 mL.
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 B-Hydroxy-B-Methylbutyrate and B- D-760 Page 
 Hydroxybutryrate Determination by HPLC using 6 of 9 
 UV/VIS Spectroscopy 
 
 8.0 Test Conditions 
 
 8.1 Gradient with wash (used for sample injections) 
 
 Time %A %B 
 
 0.00 100 0.0 
 
 15.00 80.0 20.0 
 
 15.01 0.00 100 
 
 20.00 0.00 100 
 
 20.01 100 0 
 
 32.00 100 0 
 
 8.2 Gradient no wash (used for blank and standard injections) 
 
 Time %A %B 
 
 0.00 100 0.0 
 
 15.00 80.0 20.0 
 
 15.01 100 0 
 
 19.00 100 0 
 
 8.3 Column — Acclaim C18, 5um, 120A, LC column, 250mm x 4.6mm, or equivalent
 
 8.4 Flow Rate — 0.5mL/min 
 
 8.5 UV Detection — 214nm 
 
 8.6 Injection Volume - 10uL 
 
 8.7 Column Temperature — Ambient (not controlled) 
 
 8.8 3-D Spectral Range — 200 to 300 nm 
 
 8.9 Recommended Sequence 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 
 B-Hydroxy-B-Methylbutyrate and B- D-760 2 Page 
 Hydroxybutryrate Determination by HPLC using 7 of9 
 UV/VIS Spectroscopy 
 
 8.9.1 Make at least 2 injections of a Blank (Diluent). 
 
 8.9.2 Make five injections of the Working Standard. 
 
 8.9.3 Make a single injection of each Sample Preparation. 
 
 8.9.4 Make a single injection of the Working Standard after every six samples and at
 the end of the run. 
 
 8.10 System Suitability 
 
 8.10.1 The %RSD of five consecutive injections of Working Standard is NMT 5.0%.
 
 8.10.2 The %RSD of all Working Standard injections is NMT 5%. 
 
 8.11 Column Wash and Storage 
 
 8.11.1 Rinse the column with H2O / ACN (90/10) at 1 mL/min for at least 15 min.
 
 8.11.2 Rinse the column with H2O / ACN (50/50) at 1 mL/min for at least 10 min.
 
 8.11.3 Store the column with H2O0 / ACN (50/50). 
 
 9.0 Calculations 
 
 9.) 
 0 
 % assay y 
 — 
 = 
 _ 
 — 
 R 
 Rs 
 u 
 xX - 
 Wtst 
 —V 
 q s 
 s 
 t 
 t— 
 d 
 a 
 X 
 P 
 K = 
 4 SsApl x = x 100 
 Ry Sample peak area 
 R, Mean standard peak area 
 Wtstq Weight of reference standard in mg 
 Vstq Volume of the standard preparation accounting for dilutions in mL
 P Purity of the reference standard in decimal format 
 SA Sample amount in mg (solids) or mL (liquids) 
 Vsp_ Wolume of the sample preparation accounting for dilutions in mL
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 B-Hydroxy-B-Methylbutyrate and B- D-760 
 Page 
 Hydroxybutryrate Determination by HPLC using 8 of 9 
 UV/VIS Spectroscopy 
 
 SS Serving size: Weight of a single dosage unit in mg for tablets and capsules,
 volume of a single serving from the theoretical formula in mL for liquids, or 1 for
 
 raw materials. 
 
 LA Label amount in mg per dose or 1 for raw materials 
 
 10.0 Example Chromatography 
 
 10.1 Blank 
 
 blank 
 
 § 10 141 #412 #213 +#«14 «15 4«160617~«618~«19
 Time [min] 
 
 10.2. HMB Standard 
 
 18AS053 0.5mg/mi 
 80+ 
 60+ 
 
 é 2 405 13M.B263 
 20+ 
 Don 
 _fypges.ge43s4.9 
 5 a _*Y 
 1 2 3 4 5 5 7 8 10 11 «+142 +«213 14 #2715 «16 «17 +«©18~=«19
 Time [min] 
 10.3 HMB Sample 
 200516 1.0mg/mi 
 80" 13.247 
 HMB 
 60+ 
 Z 40) 
 20+ AS g2pts 
 —_—_—— iS 
 1 2 3. «4 5 9 10 141 #«12 #«°43 144 #15 «#16 +«417~«18~«19
 Time [min] 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 
 p-Hydroxy-B-Methylbutyrate and p- D-760 2 Page 
 Hydroxybutryrate Determination by HPLC using 9 of 9 
 UV/VIS Spectroscopy 
 
 10.4 BHB Standard 
 19AS136 0.5mg/mi 
 807 
 
 604 
 2= sii S 9.863 
 20+ HB 
 a PP964-9655.350 
 1 2 3 4 5 6 7 8 O40 ad 4a AS! a4 ese Mes 7 46 S48 
 Time [min] 
 10.5 BHB Sample 
 
 200436 1.0mg/mi 
 804 
 9.H8B70 
 604 
 4 404 
 20> 
 5 ; _F-9039.949 _¥11.83912.781 
 1 2 3 4 5S 6 +7 & § 10 44 «+12 «13 «+14 «415 «16 «17 ©«18~=«19
 Time [min] 
 
 11.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/02/19 | New N/A J. Maignan Update for consistency with current methods, add recommended | - | - |
| 1 | 06/20/22 | Sequence section, replace requirements with system suitability CC- section, add example chromatography, and remove 229nm as quantitation wavelength. Remove unnecessary information and align with current SOP format, add instruction to follow test details containing product | 22-0280 | & Saseman |
| 2 | 05/15/23 | specific sample preparation, add specific sample prep CC- instructions for different dosage forms. Updated logo and format. | 23-0232 | S. Sassman |