D-789

Determination of D-Biotin by HPLC using UVVIS Spectroscopy

Section D — Laboratory Operations and Specifications Revision 2 8 pages

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1.0 Purpose Brace 16 Meah Matley. 4 Lloet-Jors,

The purpose of this procedure is to define the method for the quantification and identification of
D-Biotin in raw materials and finished products by HPLC using UV/VIS Spectroscopy.

2.0 Scope

This procedure applies to the identification and quantification of Biotin in raw material and
finished products in the QC laboratory at Ion Nutritional Labs.

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 this procedure is being followed.

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

4.1 HPLC/UV -— High pressure liquid chromatography with ultraviolet detection

4.2 QC — Quality Control

4.3 ACN - Acetonitrile

4.4 HsPO4- 85% Phosphoric acid

4.5 NaClO4— Sodium perchlorate monohydrate

4.6 K2HPO«4-— Potassium phosphate dibasic

4.7 DMSO — Dimethylsulfoxide

4.8 H20 — Water (©18.2 MQ:-cm)

[SOP

Standard Operating Procedure SOP No Rev p
Determination of D-Biotin by HPLC using UV/VIS D-789 ys ; eR
Spectroscopy

5.0 References

5.1 MV-LAB-19-143, Protocol, Validation of an Analytical Method for the Determination
of D-Biotin by HPLC/UV

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 Reference Standard

6.1.2 H20

6.1.3 ACN

6.1.4 H3PO4

6.1.5 NaClO,

6.1.6 K2HPOs,

6.1.7 DMSO

6.2 Glassware

6.2.1 Volumetric glassware as required for standard and sample preparations
6.2.2. HPLC vials, 12mm x 32mm with screw cap enclosures with septa

6.2.3. Scintillation vials

6.2.4 Mobile phase containers

6.3 Disposables

6.3.1 Centrifuge tubes

6.3.2. Plastic Luer-lock syringe

6.3.3 Nylon syringe filters, 0.45um

6.3.4 Weigh paper

[SOP

Standard Operating Procedure SOP No Rev p
Determination of D-Biotin by HPLC using UV/VIS D-789 2 3 are
Spectroscopy

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 Centrifuge

6.4.4 Adjustable Pipette

7.0 Preparation of Mobile Phase, Diluent, Standards, and Samples

7.1 Solution Preparation

7.1.1 Mobile Phase

7.1.1.1 Transfer about 0.915 g of NaClO« to a 1-L bottle, add 915 mL H20,
0.915 mL of H3PQO., and 85 mL ACN. Mix until dissolved.

7.1.2 Dhiluent

7.1.2.1. Transfer about 4.36 g of potassium phosphate dibasic to a 1-L bottle,

add 100 mL DMSO and 900 mL H20. Mix until dissolved.

7.2 Standard Preparation

7.2.1 Stock Standard: Accurately weight and transfer about 32 mg of D-Biotin
reference standard to a 100-mL volumetric flask. Dissolve in and dilute to volume

with Diluent.

7.2.2. Working Standard: Transfer 0.75 mL of Stock Standard to a 50-mL volumetric
flask. Dilute to volume with Diluent.

7.2.3 Note: Deviation from preparation above is acceptable as long as concentrations

remain within linear range.

[SOP

Standard Operating Procedure SOP No Rev p
Determination of D-Biotin by HPLC using UV/VIS D-789 2 ; ofa
Spectroscopy

7.3 Sample Preparation

7.3.1 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 below.

7.3.2 The linear range for D-Biotin is 0.25 ug/mL — 60 ug/mL. The concentration of
the sample preparation must be within the linear range.

7.3.3. For solid dose finished products: Combine and homogenize at least 10 dosage

units. Based on the weight per dose and the label amount, weigh a portion of the
homogenized sample into a suitably sized volumetric flask to generate an analyte

concentration that 1s within the validated linear range. Dilute the sample to 2/3 of

the flask volume with Diluent and shake for 20 minutes. Sonicate for 5 minutes,
and then shake again for an additional 15 minutes.

7.3.4 For liquids: use a TC pipet to transfer a portion of the sample into a suitably sized

volumetric flask to generate an analyte concentration that is within the validated
linear range. Rinse the pipet several times using Diluent, and collect the rinses in

the volumetric flask. Dilute to volume using Diluent.

7.3.5 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 homogenized sample into a suitably sized 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.

7.3.6 For raw materials: Based on the D-Biotin content, weigh a portion into a suitably
sized volumetric flask to generate an analyte concentration that is within the

validated linear range. Dilute the sample to 2/3 of the flask volume with Diluent
and shake for 20 minutes. Sonicate for 5 minutes, and then shake again for an

additional 15 minutes.

[SOP

Standard Operating Procedure SOP No Rev p
Determination of D-Biotin by HPLC using UV/VIS D-789 Z 5 see
Spectroscopy

7.3.7 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 using

Diluent to bring the analyte concentration into the linear range of measurement.

7.3.8 The final sample must be filtered or centrifuged before analyzing by HPLC.

7.3.8.1 For filtration: filter a portion through a 0.45um nylon membrane
discarding the first 2-3 mL of filtrate before collecting an aliquot for

analysis.

7.3.8.2 For centrifugation: centrifuge for 5 min at 10,000 rpm.

8.0 Test Conditions

8.1 HPLC Parameters

8.1.1 Column: YMC-Pack Pro C8, 3 um, 4.6 mm x 150 mm

8.1.2 Column Temperature: 30 °C

8.1.3. Flowrate: 1.2 mL/min
8.1.4 Wavelength: 200 nm

8.1.5 Injection Volume: 50 uwL

8.1.6 Run Time: at least 20 minutes

8.1.7 Recommended Spectral Range (for Identification)- 200 nm to 600 nm

8.2 Recommended Sequence

8.2.1 Make at least 2 injections of the diluent.

8.2.2 Make five (5) injections of Standard Solution.

8.2.3 Make a single injection of each Sample Preparation.

8.2.4 Make a single injection of the Standard Solution after every ten (10) sample
injections or at the end of a run.

8.3 System Suitability Requirements

8.3.1 The %RSD of the first five (5) standard injections is NMT 2.0%.

8.3.2. The %RSD of all standard injections is NMT 2%.

[SOP

Standard Operating Procedure SOP No Rev
Determination of D-Biotin by HPLC using UV/VIS D-789 2 : see
Spectroscopy

8.3.3. The average (n=5) tailing factor is NMT 1.5.

8.4 Retention Times
8.4.1 D-Biotin: about 16 min

8.5 Recommended System Setup

8.5.1 Purge channel A with H2O/methanol (90/10) at 5 mL/min for at least 5 min.

8.5.2 Rinse the column with H2O/methanol (90/10) at 1 mL/min for at least 5 min.

8.5.3. Purge channel A with Mobile Phase at 5 mL/min for at least 5 min.

8.5.4 Begin flow of Mobile Phase through the column at 1 mL/min.

8.6 | Column Wash and Storage

8.6.1 Rinse the column with H2O / ACN (90/10)

8.6.2 Store the column in H20 / ACN (50/50)

9.0 Calculations

Ry ., WtstqxP .. SS — Vspi
9.1 assay = 9 XX aX 7" x 100
Ry Sample peak area

Rg Mean standard peak area

Wtsta Weight of the reference standard in mg

Vsta 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)

SS Serving size: Average weight of ten dosage units in mg for tablets and

capsules, volume of a single serving from the theoretical formula in mL for
liquids, or 1 for raw materials.

Vent Volume of the sample preparation accounting for dilutions in mL

LA Label amount in mg (use | for raw materials)

[SOP

Standard Operating Procedure SOP No Rev p
Determination of D-Biotin by HPLC using UV/VIS D-789 z 7 “
Spectroscopy

10.0 Example Chromatography and Spectrum
10.1 Blank

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Time [min]
10.2. Working Standard
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Time [min]

10.3. Finished Product Sample
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Time [min]

[SOP

Standard Operating Procedure SOP No Rev
Page
Determination of D-Biotin by HPLC using UV/VIS D-789
8 of 8
Spectroscopy

10.4 Biotin UV Spectrum

; Biotin - 17.155 (2023-01-27 15-55-18-05-00-03-r003.dx)

90-
80-
70-
60-

40-
30
20-
10-
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
Am

11.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 02/11/20 | New N/A S. Sassman Minor edits for consistency with current methods, add instruction l 04/10/23 to follow product specific test details, add example CC-23-0176 S.Sassman chromatography, add recommended system start up and shut down. | - | - |
| 2 | 08/02/24 | Added note to allow flexibility in standard prep. CC- | 24-0353 | D. Hashmi |
| 3 | 10/15/25 | Added note to allow for preparations to be anywhere inside the CC- linear range of Biotin | 25-0421 | M. Autrey |