D-1014

Determination of Paraxanthine in Raw Materials & Finished Products by HPLC-UV

Section D — Laboratory Operations and Specifications 8 pages

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1.0 Purpose

This document describes the analytical procedure for the determination of Paraxanthine in raw

materials and finished products.

2.0 Scope

This procedure applies to the identification and quantification of Paraxanthine in raw materials

and finished products. This method was validated under protocol PRTCL-22-0052.

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 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 Management and/or Analytical Development

Personnel to keep this procedure current with the associated monographs and laboratory
practices.

4.0 Definitions

4.1 QC — Quality Control

4.2 ACN — Acetonitrile

4.3 ACS — American Chemical Society

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Standard Operating Procedure SOP No | Rev
Determination of Paraxanthine in Raw Materials & D-1014 0 Page 2 of 8
Finished Products by HPLC-UV

4.4 HPLC — High Performance Liquid Chromatography

45 UV-Vis — Ultraviolet-Visible (Detection)

5.0 References

5.1 PRTCL-22-0052, Protocol, Validation of an Analytical Method for the Determination
of Paraxanthine by HPLC-UV

6.0 Supplies

6.1 Chemicals — All reagents are ACS grade or better

6.1.1 Milli-Q Water

6.1.2 ACN

6.1.3 Paraxanthine 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

6.2.3. Weigh paper

6.2.4 Syringes with 0.45u nylon filters

6.3. Equipment

6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven

and UV-Vis detector with a chromatographic data handling system

6.3.2 Analytical Balance

6.3.3. Sonicator Bath

[SOP D-101 | Page 3 of 8]

Standard Operating Procedure SOP No Rev
Determination of Paraxanthine in Raw Materials & D-1014 Page 3 of 8
Finished Products by HPLC-UV

6.3.4 Wrist Action Shaker

7.0 Procedure

7.1 Mobile Phase & Diluent Preparation

7.1.1. Mobile Phase

7.1.1.1 Mobile Phase A = Water

7.1.1.2 Mobile Phase B = ACN

7.1.2 Extraction Solvent / Diluent

7.1.2.1 Water

7.2 Standard Prep

7.2.1. Prepare standard stock solution at 0.2 mg/ml in water. Sonicate for 10 minutes
to ensure dissolution, then shake vigorously. Cool to room temperature, then

dilute 1:10 with diluent. Shake vigorously then filter a Sml aliquot for analysis,
discarding the first 3-4ml of filtrate.

7.2.2 Alternative standard preparations are acceptable as long as the preparations are
within the linear range of this method.

7.3 Sample Preparation

7.3.1 Specific sample testing details are provided in each products profile. If a

specific details section is not available then follow preparation procedure as

described below, maintaining concentration within the linear range of this
method.

7.3.2 The validated linear range for the analytical method is 0.01284 — 0.02996
mg/ml. Prepare raw material samples like standards.

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Standard Operating Procedure SOP No Rev
Determination of Paraxanthine in Raw Materials & D-1014 Page 4 of 8
Finished Products by HPLC-UV

7.3.3 Prepare finished product sample stock solution at 0.2 mg/ml in water (based on
the finished product profile). Shake mechanically at ~ half volume for 15

minutes then QS to volume. Sonicate for 10 minutes then shake vigorously.
Dilute 1:10 with diluent and shake vigorously. Filter a Sml aliquot for analysis,

discarding the first 3-4ml of filtrate.

7.4 HPLC Parameters

7.4.1 Column: Phenomenex Kinetex XB-Cig, 4.6 x 150mm, 2.6um SPP (Or

Equivalent)

7.4.2 Column Temperature: 40°C

7.4.3. Flow rate: 1.0 mL/min

7.4.4 Mobile Phase Gradient:

Time,min | %A | %B
0 97 3
2 97 3
10 84 16
12 50 50

12.1 97 3
15 oF 3

7.4.5 Wavelength: 271 nm

7.4.6 Injection Volume: 5 wL

7.4.7 Run Time: 15 minutes

7.4.8 Recommended 3-D Spectral Range (for Identification): 210nm - 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 the Working Standard.

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Standard Operating Procedure SOP No Rev
Determination of Paraxanthine in Raw Materials & D-1014 0 Page 5 of 8
Finished Products by HPLC-UV

7.5.3. Make a single injection of each Sample Preparation.

7.5.4 Make a single injection of the Working Standard after every ten (10) sample

injections and/or at the end of the run.

7.6 System Suitability Requirements

7.6.1 The %RSD of five (5) consecutive standard injections is NMT 2%.

7.6.2 The %RSD of all standard injections is NMT 3%.

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 % assay:

1.7.1 % = <2 x si x ~zl x 10x SS0/LA

R., Sample peak area

R, Mean (n=5) standard peak area

Wt..4 Weight of the reference standard, mg

Vora Volume of the standard preparation accounting for dilutions, mL

P Purity of the reference standard in decimal format

SA Sample amount, mg

Vin Volume of the sample preparation accounting for dilutions, mL

SS Use average dosage weight for solids or use serving size specified in
Product Profile for powders and liquids. Use “1” for raw materials

LA Label Amount (Use 1 for Raw Materials)

7.8 System Wash, Column Wash and Column Storage

7.8.1 Wash and store the column in 75:25 ACN / Water

[SOP D-101 | Page 6 of 8]

Standard Operating Procedure SOP No Rev
Determination of Paraxanthine in Raw Materials & D-1014
Page 6 of 8
Finished Products by HPLC-UV

8.0 Chromatograms

8.1 Typical Diluent Chromatogram

3 Pox RnBBD ter UMS 427i nm

as
ae

Q40-

8.2 Typical Standard Chromatogram

3 RrxRnBS2B vs U/VS1WLZ7irm
3) nf 1-Paax- 807

Co Tr yO De Pe ee ee ye pe ee Ce ee oe UU See ES DT ta ee EL ae ee

[SOP D-101 | Page 7 of 8]

Standard Operating Procedure SOP No | Rev
Determination of Paraxanthine in Raw Materials & D-1014 0 Page 7 of 8
Finished Products by HPLC-UV

8.3. Typical Raw Material Chromatogram

an RFKRNBB2B RMI WAS1VWLZInm
inf
04 1-Paax-8047
00}

504

20!

20-
60

40:

eer er ee Oe a a rr a a Sr a a ar

8.4 Typical Finished Product Chromatogram

ay, 1 PrxRnBSZHt PR US IWLZrm
— néU

ao 2-Pearax-806D

00!

no
{ | 7 aie Pele cs ] — ao |

‘Sf ke ae eS oe ee a nr

[SOP D-101 | Page 8 of 8]

Standard Operating Procedure SOP No | Rev
Determination of Paraxanthine in Raw Materials & D-1014 0 Page 8 of 8
Finished Products by HPLC-UV

9.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 11/10/22 | New procedure. N/A C. Perry | - | - |