D-743

Coenzyme Q10 (Ubidecarenone) Determination by HPLC using UV-Vis Detection

Section D — Laboratory Operations and Specifications Revision 2 7 pages

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1.0 Purpose

The purpose of this procedure is to define the method for the quantitative analysis and/or

identification of Coenzyme Q10 and its acid form in raw materials and finished products using

HPLC with UV/VIS detection.

2.0 Scope

This procedure applies to the quantification and identification of Coenzyme Q10. Some

excipients and dietary ingredients used in finished products can interfere with the analysis of
Coenzyme Q10.

3.0 Responsibility

3.1 It is the responsibility of QC and Analytical Chemists to follow this procedure.

3.2 ‘It is the responsibility of QC Laboratory Management to implement this procedure and

to ensure that the procedure is being followed.

3.3. ‘It is the responsibility of QC Laboratory and Analytical Development Management to

keep this procedure current with latest lon Labs practices.

4.0 Definitions

4.1 HPLC — High Performance Liquid Chromatography

42 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums

4.3 Ubidecarenone — Coenzyme Q10

4.4 FeClh — Ferric Chloride

4.5 HO - Water (> 18.2 MQ:cm)

4.6 CofA — Certificate of Analysis

[SOP

Standard Operating Procedure SOP No Rev
Coenzyme Q10 (Ubidecarenone) Determination by D-743 Page
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HPLC using UV/VIS Detection

5.0 References

5.1 | Phenomenex, Application ID# 1070

5.2 Determination of Coenzyme Q10 Content in Raw material and Dietary Supplements by

High-Performnce Liquid chromaography-UV: Collaborative Study. J AOAC Int. 2008;
91(4): 702-708

5.3. MV-LAB-15-035, Protocol, Coenzyme Q10 (Ubidecarenone) Determination using

HPLC coupled with UV/VIS Spectroscopy

6.0 Reagents, Supplies, Glassware and Equipment

6.1 Chemicals: All reagents are HPLC grade or better.

6.1.1 Millipore Water

6.1.2 Denatured Ethanol

6.1.3. Methanol

6.1.4 FeCls

6.1.5 H20

6.2 Glassware and Supplies
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 50mL Volumetric Flask

6.2.5 100mL Volumetric Flask

6.3. Disposables

6.3.1 10mL Pipette Tips

6.3.2 ImL Pipette Tips

6.3.3 200uL Pipette Tips

6.3.4 16mL Test Tubes

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Standard Operating Procedure SOP No | Rev
Coenzyme Q10 (Ubidecarenone) Determination by D-743 2
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HPLC using UV/VIS Detection
6.3.5 Disposable Plastic Luer Lock Syringe —- 3mL, 6mL, or 10mL

6.3.6 Nylon Syringe Filters, 0.45um

6.3.7 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 Stir Plate

6.4.4 Wrist Action Shaker

6.4.5 Vortex

6.4.6 Sonicator Bath

6.4.7 200uL, ImL, and 10mL Pipettes- adjustable

6.4.8 Eppendorf Centrifuge

7.0 Preparation of Mobile Phase, Diluent, Samples and Standards

se Mobile Phase A — Ethanol

7.2 Mobile Phase B — Methanol

im Diluent — 1% FeCl; in Ethanol

7.3.1 Add 1.0 g of FeCh to 1000mL ofEthanol.

7.3.2 Sonicate for 30min to dissolve.

7.3.3. Scale as necessary.
7.4 Standard Preparation

7.4.1 The linear range of the method is 0.0025 mg/mL — 0.25 mg/mL. The standard and

sample preparations must be within the linear range of the method.

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7.4.2 Use the actual purity from the CofA or the standard certification for Coenzyme
Q10 reference material for calculations. The stock standard preparation reflects

100% content for the analyte assayed.

7.4.3. The standard is prepared by weighing no less than the minimum weight of the

analytical balance, then suspended in the final volume and sonicated for 25
minutes. Then the sample is allowed to cool to RT before use.

7.4.4 Dilutions are prepared using diluent. Dilutions can be made using volumetric

flasks or using 10mL, lmL, and 200uL variable 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.

7.5 Sample Preparation

7.5.1 At least 10 dosage units are pooled and/or ground by mortar and pestle as

necessary.
7.5.2 Based on the fill weight, tablet weight, or raw material potency, weigh a portion

of the pooled dosages to generate an analyte concentration that is within the
validated linearity and solubility range.

7.5.3. Samples can be dissolved in diluent at any volume starting from 10mL and any

weight greater than the minimum weight of the analytical balance.

7.5.4 The sample is suspended in the final volume and sonicated for 25 minutes. Then

the sample is allowed to cool to RT before use.

7.5.5 Before injection, insoluble matter should be removed via filtration using a nylon
syringe filter. Discard at least 2mL of the filtrate before collecting a portion in an

HPLC vial for analysis.

7.5.5.1. Alternatively, samples and standards can also be centrifuged at 5000
RPM for 3 minutes in an Eppendorf centrifuge to pellet insoluble matter.

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Standard Operating Procedure SOP No | Rev

Coenzyme Q10 (Ubidecarenone) Determination by D-743 2 Page
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HPLC using UV/VIS Detection
7.5.6 For Coenzyme Q10 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 Gradient — Isocratic

Time YA %B Gradient Type

0.00 65 35 0

15.0 65 35 0

8.2 Column — Luna C18 (2), 5um, 100A, LC column, 150mm x 4.6mm or equivalent

8.3 Flow Rate — 1.0mL/min

8.4 UV Detection —-275nm

8.5 Injection Volume - 20u.L

8.6 Column Temperature — 28°C

8.7 Retention Time — about 11 min

8.8 Recommended Sequence

8.8.1 Make at least 2 injections of the diluent.

8.8.2 Make five (5) injections of Standard Solution.

8.8.3 Make a single injection of each Sample Preparation.

8.8.4 Make a single injection of the Standard Solution after every six (6) samples and
at the end of the run.

8.9 System Suitability Requirements

8.9.1 The %RSD of the first five (5) standard injections is NMT 5.0%.

8.9.2 The %RSD of all standard injections is NMT 5%.

8.10 Column Wash and Storage

8.10.1 Column wash is not required.

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Standard Operating Procedure SOP No | Rev
Coenzyme Q10 (Ubidecarenone) Determination by D-743 : : ey
HPLC using UV/VIS Detection °

8.10.2 Store the column with mobile phase.

8.11 Example calculations for determining finished product % label or raw material % purity

9 we Bu yy WhetaXP | Uspt y SS
8.11.1 % assay 7 x Vea Xe 100

ay Sample peak area

R, Mean standard peak area

Wtstq Weight of reference standard in mg

Vista 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)

Vent Volume of the sample preparation accounting for dilutions in mL

55 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 | for raw materials

8.12 Example Chromatography

8.12.1 Blank

DAD1A,Sig=275.04.0 Ref=360.0,100.0
80+ <

_, 60 .
= 40: i

207 |

1 2 3 4 5 6 7 8 9 1011 12 13 14 ~°«168
Time [min]

[SOP

Standard Operating Procedure SOP No | Rev
Coenzyme Q10 (Ubidecarenone) Determination by D-743 2 :
. ‘ 0
HPLC using UV/VIS Detection
8.12.2 Standard

q =O
40-
20- «

12 9 4° RB 8 FB oe 4 at aR a ta
Time [min]

8.12.3 Sample

1 2 3 4 5S 6 7 8&8 9 4 44 12 13 14 «15
Time [min]

9.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 02/05/16 | New I 01/29/19 Scheduled review: updated responsibilities and fixed typos. 19-0107 J. Maignan Update to reflect current practices, add linear range, add Zz 04/15/22 recommended sequence, add system suitability section, add column = CC-22-0181 S. Sassman wash and storage, add example chromatography. | 16-0104 | X. Shao |