D-740
Monacolin K 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 trace analysis and/or identification
of monacolin K and its acid form in raw materials and dietary supplements using HPLC and
UV/VIS spectrophotometry.
2.0 Scope
This procedure applies to the quantification and identification of monacolin K and its acid form.
This procedure applies to the identification of monacolin K forms in raw materials and finished
products using spectral analysis and quantitation of monacolins using monacolin K as the
standard.
3.0 Responsibility
3.1 It is the responsibility of QC Chemists to understand and work within the guidelines of
this procedure.
3.2 It is the responsibility of QC Laboratory Management to ensure compliance with this
procedure.
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.] HPLC — High Performance Liquid Chromatography
4.2 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums
4.3 Lovastatin — Monacolin K
4.4 H3PQ4— Phosphoric Acid
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Monacolin K Determination by HPLC using UV/VIS D-740 Page
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Spectroscopy
4.5 NaOH — Sodium Hydroxide
4.6 ACN — Acetonitrile
4.7 CofA — Certificate of Analysis
4.8 H20O — Water
5.0 References
5.1 MV-LAB-14-002, Protocol, Monocalin K Determination by HPLC
D2 Lovastatin USP Monograph
6.0 Supplies
6.1 Chemicals: All reagents are HPLC grade or better.
6.1.1 H2O(e 18.2 MQ-cm)
6.1.2 ACN
6.1.3 H3PQO,
6.1.4 NaOH
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. 1L Mobile Phase Container
6.2.4 Volumetric Flasks
6.2.5 Volumetric Pipets
6.3 Disposables
6.3.1 200uL, lmL and 10mL Pipette Tips
6.3.2 16mL Test Tubes
6.3.3. Disposable Plastic Luer Lock Syringe — 3mL, 6mL, or 10mL
6.3.4 Nylon Syringe Filters, 0.45um
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Standard Operating Procedure SOP No | Rev
Monacolin K Determination by HPLC using UV/VIS | 0-740 3 : oes
Spectroscopy
6.3.5 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. Vortex
6.4.4 Stir Plate
6.4.5 Eppendorf Micro-Centrifuge
6.4.6 200uL, 1mL and 10mL Pipettes
7.0 Preparation of Mobile Phase, Diluent, Samples, and Standards
7.1 Mobile Phase A (0.1% H3POg4 in H20)
7.1.1. Transfer 1000 mL of H20 to a mobile phase bottle.
7.1.2 Add 1.0 mL of H3PO«, and mix well.
7.2. Mobile Phase B (100% ACN)
7.3 Diluent (MeOQH:H20:H3PO04 775:225:0.225)
7.3.1. Transfer 775mL of MeOH to a mobile phase bottle.
7.3.2 Add 225mL of H20.
7.3.3. Add 225 uwL of H3PO4, and mix well.
7.4 Stock Standard Preparation
7.4.1. Accurately weigh and transfer about 25 mg of reference material into a 100-mL
volumetric flask.
7.4.2 Dilute to volume with Diluent.
7.4.3. Sonicate for 10 minutes to dissolve.
7.5 Working Standard Preparation
Be Pe i Transfer 2.0 mL of the Stock Standard to a 50-mL volumetric flask.
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Standard Operating Procedure SOP No | Rev
Monacolin K Determination by HPLC using UV/VIS D-740 3 —
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7.5.2 Dilute to volume with Diluent, and mix well.
7.6 Sample Preparation
7.6.1 The linear range of the method is 1 ug/mL — 30 ug/mL. The analyte
concentration in all working sample preparations must be within this range.
7.6.2 Samples can be dissolved in Diluent at any volume starting from 50mL and any
weight > 50mg.
7.6.3. The sample is suspended in one third the final volume and sonicated for four
minutes. The sample is cooled to room temperature before bringing to volume.
At volume, the sample is sonicated for an additional six minutes and allowed to
cool to room temperature before use.
7.6.4 Before injection, insoluble matter should be removed via filtration using a nylon
syringe filter. Discard at least 0.5mL of the sample before collecting filtrate.
Dilute filtrate as needed then add 1mL of the final dilution to an HPLC vial for
analysis.
7.6.4.1 Alternatively, samples and standards can also be centrifuged at 6000
RPM for 5 minutes in an Eppendorf centrifuge to pellet insoluble
matter.
7.6.5 For non-Red Yeast Rice raw materials being analyzed for the first time using
this method, in-process verification is required to demonstrate spectral purity
and extraction efficiency before the method can be implemented.
8.0 Test Conditions
8.1 Isocratic
Time YA %B Gradient Type
0.00 35 65 0
12.00 35 65 )
8.2 Column — Luna C18 (2), 5um, 100A, LC column, 150mm x 4.6mm or equivalent.
8.3 Flow Rate — 1.0mL/min
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Standard Operating Procedure SOP No | Rev
Monacolin K Determination by HPLC using UV/VIS D-740 3 : aes
Spectroscopy
8.4 UV Detection —238nm
8.5 3-D Spectral Range — 200nm to 300nm
8.6 Injection Volume - 20uL
8.7. Column Temperature — 40°C
8.8 | Recommended Sequence
8.8.1 | Make at least 2 injections of a Blank (Diluent).
8.8.2 Make five injections of the Working Standard.
8.8.3. Make a single injection of each Sample Preparation.
8.8.4 Make a single injection of the Working Standard after every six samples and at
the end of the run.
8.9 System Suitability
8.9.1 The %RSD of five consecutive injections of Working Standard is NMT 5.0%.
8.9.2 The %RSD of all Working Standard injections is NMT 5%.
8.10 Column Wash and Storage
8.10.1 Rinse the column with H2O / ACN (50/50) at 1 mL/min for at least 15 min.
8.10.2 Store the column with H2O / ACN (50/50).
9.0 Example Calculation
% assay = 2Ru~ X WtstaXP -, Vspl x= x 100
9.1 Fa Peak area of monacolin peak in the Sample Preparation
Re Mean standard peak area
Wt.q 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)
[SOP
Standard Operating Procedure SOP No Rev
Monacolin K Determination by HPLC using UV/VIS D-740
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Spectroscopy
10.0 80-
70-
60-
507
40-
30-
207
10-
UAm
100-
80-
60- UAm
Vent Volume of the sample preparation accounting for dilutions in mL
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.
9.2 Total monacolins are calculated by summing all peaks that were determined to be
monacolins by retention time and/or spectral match.
Example Chromatography
10.1. Blank
DILUENT
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Time [min]
10.2. Working Standard
21AS003 0.02MG/ML
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Time [min]
[SOP
Standard Operating Procedure SOP No | Rev
Monacolin K Determination by HPLC using UV/VIS D-740 3 . ney
0
Spectroscopy
10.3. Sample
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Time [min]
11.0 Revision History
| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 05/07/14 | New ] 09/16/16 Biennial review: Updated SOP format. 16-0849 N. Zhang | 14-0402 | B. Johns |
| 5 | 11/26/19 | 3 Year review — Updated to be in line with current procedure and practices. Update for consistency with current methods and lab practices, | 19-0903 | W. Gonzales |
| 3 | 08/05/22 | add recommended sequence section, replace requirements with CC- system suitability section, add column wash and storage, add example chromatography | 22-0342 | S. Sassman |