Monacolin K Determination by HPLC with UVVis Spectroscopy

Original Document

Scanned document (image-only PDF)

Extracted Text

Searchable text extracted from PDF

1.0 Purpose 
 
 This document describes the analytical procedure for the determination of Monacolin K (MK)
 in raw materials and finished products. 
 2.0 Scope 
 
 This procedure applies to the identification and quantification of Monacolin K in raw materials
 
 and finished products. Monacolin K can be present in two forms: (1) the unhydrolyzed form
 interchangeably referred to as, among many other synonyms: Monacolin K, Monacolin K
 Lactone and Lovastatin, and (2) the hydrolyzed acid form referred to as Monacolin K Acid.
 Monacolin K Acid may or may not be present. If Monacolin K Acid is present, its result is
 expressed as equivalents Monacolin K Lactone. MK (Total) may also be reported, and is
 
 expressed as the sum of Monacolin K and Monacolin K Acid. This method was validated
 under Protocol PRTCL-20-0029 and reported in RPT-20-0034. 
 
 3.0 Responsibility 
 3.1 It is the responsibility of QC and Analytical chemists who have verified their ability to
 
 execute this procedure to follow this procedure. 
 3.2 It is the responsibility of the QC Laboratory Management to implement this procedure and
 
 to ensure that the procedure is being followed. 
 3.3 It is the responsibility of the QC Laboratory Management and AD Personnel to keep this
 
 procedure current with the associated monographs and laboratory practices.
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2 AD - Analytical Development 
 4.3 MK — Monacolin K 
 « 
 4.4 ACN — Acetonitrile 
 
 4.5 MeOH — Methanol 
 4.6 H3P0O4— Phosphoric Acid 
 
 
 

[SOP 

 Standard Operating Procedure 
 Monacolin K Determination by HPLC with UV/Vis SOP No Rev 
 D-764 0 Page 2 of 6 
 Spectroscopy 
 4.7 NaOH — Sodium Hydroxide 
 
 4.8 ACS — American Chemical Society 
 4.9 HPLC — High Performance Liquid Chromatography 
 
 4.10 UV/Vis — Ultraviolet & Visible Electromagnetic Spectra 
 5.0 References 
 
 5.1 PRTCL-20-0029 — Monacolin K Determination by HPLC Using UV/Vis Spectroscopy
 
 5.2 RPT-20-0034 — Monacolin K Determination by HPLC Using UV/Vis Spectroscopy
 6.0 Supplies 
 
 6.1 Chemicals — All reagents are ACS grade or better. 
 
 6.1.1 Milli-Q Water 
 
 6.1.3 ACN 
 6.1.4 H3P04 
 
 6.1.55 IN NaOH 
 6.1.6 MeOH 
 
 6.1.7 MK (Lovastatin) Reference Standard 
 6.2 Supplies and Glassware 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 6.2.2 Volumetric glassware and/or adjustable Pipettes and tips 
 6.2.3. Weigh paper and/or funnels 
 
 6.2.4 Syringes with 0.45 Nylon Syringe Filters 
 
 6.3 Equipment 
 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column
 
 oven and UV detector with a chromatographic data handling system
 6.3.2 Analytical Balance 
 
 6.3.3 Sonicator 
 
 6.3.4 pH Meter 
 
 7.0 Procedure 
 
 7.1 Mobile Phase & Diluent Preparation 
 7.1.1 | Mobile Phase 
 
 

[SOP 

 Standard Operating Procedure 
 Monacolin K Determination by HPLC with UV/Vis S 
 D 
 O 
 P 
 7 6 
 N 
 4 
 o Re 
 0 
 v 
 Page 3 of 6 
 Spectroscopy 
 7.1.1.1 | Mobile Phase A: Add 1000 wL of H3P04 to 1000 mL of water and
 mix well. 
 7.1.1.2 Mobile Phase B: ACN 
 7.1.2 Extraction Solvent / Diluent 
 7.1.2.1 pH 4.0 Buffer: Add 950 uL of H3PO4 to 950 mL of water and mix
 well, then pH to 4.0 with IN NaOH. 
 7.1.2.2 Diluent / Extraction Solvent: Add 250 mL of the pH 4.0 Buffer to
 750 mL of MeOH and mix well. 
 7.1.3 Preparations may be scaled as necessary 
 7.2 Standard Preparation 
 7.2.1 Accurately weigh and transfer about 25 mg Lovastatin reference standard into
 a 100 mL volumetric flask. Add ~50 mL of Diluent and sonicate for five
 minutes. Add ~ 45 mL of Diluent and sonicate for an additional five minutes.
 7.2.2 Cool to ambient then QS to volume with Diluent and mix well — this is the
 Lovastatin Stock. Dilute the Lovastatin Stock 4:100 with Diluent — this is the
 Lovastatin Working Standard. 
 7.3, Sample Preparation 
 7.3.1 The validated range for the analytical method is 0.17 — 10.86 g/mL.
 7.3.2 For finished products, extract sufficient sample with Diluent in order to
 
 generate an MK concentration that is within the validated linear range.
 7.3.3 Prepare raw materials like standards, remembering that red yeast rice powders
 generally contain approximately 0.2% MK (Total). 
 
 7.3.4 Finished product samples can be dissolved in Diluent at any volume starting
 from 100mL. The volume chosen must be in the solubility range of MK
 
 (validated at approximately 16 pg/ml). To manage large volumes, the sample
 can be initially dissolved in a smaller volume that is within the solubility range
 and a portion further diluted to bring the MK concentration into the linear
 range. 
 
 7.3.5 Fill the flask to about 50% of the chosen volume with Diluent and sonicate for
 5 minutes. Then fill the flask to about 95% of the chosen volume with Diluent
 and sonicate for an additional 5 minutes. Cool to ambient then QS to volume
 
 with Diluent. 
 7.3.6 Perform further dilutions as required using Diluent. Filter a 5ml aliquot for
 analysis, discarding the first 3-4ml of filtrate. 
 
 

[SOP 

 Standard Operating Procedure 
 Monacolin K Determination by HPLC with UV/Vis S 
 D 
 O 
 P 
 76 
 N 
 4 
 o Re 
 0 
 v 
 Page 4 of 6 
 Spectroscopy 
 7.3.7 For finished products or raw materials being analyzed for the first time using
 this method, an in process validation is required to demonstrate spectral purity,
 baseline separation of peaks, and extraction efficiency as a part of system
 suitability before data can be reported using this method. 
 7.4 HPLC Parameters 
 7.4.1 Column: Waters Cortecs Shield RP18, 4.6 x 130mm, 2.7m (Or Equivalent)
 7.4.2 Column Temperature: 40°C 
 7.4.3 Flow rate: 1.0 mL/min 
 7.4.4 Mobile Phase: Isocratic 55% A / 45% B 
 7.4.5 Wavelength: 239 nm 
 7.4.6 Injection Volume: 10 pL [LOQ = 0.17 g/ml. For peaks presenting with S/N
 <10, report as < (0.17 g/ml / Sample Conc in ug/ml) * 100%.)
 7.4.7 Run Time: 20 minutes 
 74.8 3-D Spectral Range (for Identification) - 210nm to 350nm 
 7.5 Recommended Sequence 
 7.5.1 Make at least 2 injections of the Diluent. 
 7.5.2 Make at least five (5) injections of MK Working Standard. 
 7.5.3 Make a single injection of each Sample Preparation. 
 
 7.5.4 Make a single injection of the Standard Solution after every ten (10) sample
 injections and/or at the end of a run. 
 7.6 System Suitability Requirements 
 
 7.6.1 The %RSD of five (5) consecutive standard injections is NMT 2.0%
 
 7.6.2 The %RSD of all standard injections is NMT 3.0%. 
 
 7.6.3 If present, any interference in the diluent should be subtracted out of the
 sample and standard peak areas. 
 7.7 Example calculations for determining % Analyte: 
 
 7.7.1 % MK (Lactone) = — R R. u y Wt V s e t a ax P * | V “s spai * 100
 
 Ruy Sample peak area 
 [Note: To calculate % MK Acid, use the area of the Acid peak. To
 
 calculate % MK (Total), add the peak areas due to both the Acid (if
 present) + Lactone forms.] 
 
 

[SOP 

 Standard Operating Procedure 
 Monacolin K Determination by HPLC with UV/Vis 4hy — Page 5 of 6 
 Spectroscopy 
 
 R, Mean (n=5) standard peak area 
 
 Wtoia Weight of the reference standard 
 
 Vsta Volume of the standard preparation accounting for dilutions in mL
 P Purity of the reference standard in decimal format 
 
 SA Sample amount 
 
 Vspt Volume of the sample preparation accounting for dilutions in mL
 
 7.8 System Wash, Column Wash and Column Storage 
 7.8.1 Wash and store the column in 50:50 ACN / Water. 
 
 8.0 Chromatograms 
 
 8.1 Typical Diluent Chromatogram 
 
 42.0000 3 
 m A 
 Mo 
 U 
 nacolin K Run 05-11-20 #2 Diluent 
 UV_VIS_1 WVL:239 nm
 1.754 
 1.50 
 1.254 
 1.00+ 
 0.75 5 
 0.50+ 
 0.0 1q.T 0 bl 2.T 0 3.qT 0 * 4T0 . 5.LU 0 6q.T 0 7.qT 0 ¥ 8.qT 0 9.qT 0 10T. 0 11'. 0 12qT.0 15T. 0 16a.0m : 17n.0d 18—.0 19—.0 20.0
 8.2 Typical Working Standard Chromatogram 
 
 20.0 3 Monacolin K Run 05-11-20 #3 WSTD 
 mAU UV_VIS_1 WVL:239 nm
 18.04 1-Monacolin K - 14.580 
 16.04 
 14.04 
 12.04 
 10.0 4 
 8.0- 
 6.0- 
 
 A a a ae a ee a er 
 
 

[SOP 

 Standard Operating Procedure <apiN . 
 Monacolie n K Determien atie on by HPLC wiet h UV/Vies D-764O o 0e v Page 6 of 6
 Spectroscopy 
 
 8.3. Typical Raw Material Chromatogram 
 
 400 3 m A M U onacolK iRunn 05-11-2#09 RM-1 UV_VIS_1 WVL:239 nm
 
 3305..0044 | 
 
25.04 &

20.04 ge

 e 
 15.04 6 
10.04 Ra

 ie 
 5.04 Ee 
 ge 
 9 
 Ky R ge & on 
 jt j i on | : ae 
 | 
 | ‘iene 
 ‘nn Soi MM A ag ae, eg A ane eee 90 OSCOSCSSCSSCC “10 80S*S*~«SSS 
 8.4 Typical Finished Product Chromatogram 
 7 7 MoreolnK Run 6-1-2081 FP WW.VIS-4 W239 
 “Wnty 
 ‘04 
 
 i 
 1 1-MonacAocild i- 7n72K3 
 
 iD 
 MonaK c«14o60 
 4-109 
 04-—_J | ie iy Y~ 
 
 | mn 
 oo a 0 oO 8 8 Db 8 © nh W Hw Ww a a er er ee 
 
 9.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 04/14/21 | New N/A C. Perry | - | - |