D-719

Amino Acid Determination using OPA and FMOC Derivitivization coupled with HPLC and UV-Vis

Section D — Laboratory Operations and Specifications Revision 4 9 pages

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1.0 Purpose

The purpose of this procedure is to describe a method for the quantitative analysis of amino acids

in finished products and raw materials using derivitivization coupled with HPLC and UV/VIS
spectrophotometry.

2.0 Scope

This procedure is applicable to the analysis of B-alanine, aspartic acid, carnosine, glutamine,

isoleucine, leucine, lysine, phenylalanine, taurine, theanine, threonine, tyrosine, and valine using
HPLC in the QC laboratory at Ion Labs.

3.0 Responsibility

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

3.2 It is the responsibility 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 aligned with current practices.

4.0 Definitions

4.1 ACN — Acetonitrile

4.2. |NaH2PO4— Sodium phosphate monobasic

4.3. HPLC — High Performance Liquid Chromatography

4.4 OPA — Ortho-phthalaldehyde

4.5 Na2BsO7.10H20 — Sodium tetraborate decahydrate

4.6 NaOH — Sodium Hydroxide

4.7 HoO- Water, =>18.2MQ-cm, 0.22um filtered

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4.8 QC — Quality Control

5.0 References

5.1 Henderson JW, Ricker RD, Bidlingmeyer BA, Woodward C. Rapid, Accurate, Sensitive,

and Reproducible HPLC Analysis of Amino Acids. Agilent Technologies. 2000.

5.2 MV-LAB-13-062, Protocol, Amino Acid Determination using Derivitivization and
HPLC with UV Spectroscopy

5.3 MV-LAB-14-055, Protocol, L-Theanine and f-alanine Determination using OPA

Derivitivization coupled with HPLC and UV/VIS Spectroscopy

5.4 MV-LAB-18-008, Protocol, Glutamine, Threonine, Valine, Phenylalanine, Isoleucine,
Lysine and Aspartic acid Determination using OPA Derivitization coupled with HPLC

and UV/Vis Spectroscopy

5.5 MV-LAB-18-214, Protocol, Carnosine Determinations using OPA Derivitization
coupled with HPLC and UV/Vis Spectroscopy.

6.0 Procedure

6.1 Reagents: all reagents are HPLC grade or better.
6.1.1 Reference standards for target analytes

6.1.2 H20

6.1.3. ACN

6.1.4 NaH2PQ,

6.1.5 Methanol

6.1.6 NaOH

6.1.7. OPA - Agilent Part Number 5061-3335

6.1.8 2-Mercaptoethanol
6.1.9 Na2B407.10H20

6.2 Supplies and Glassware

6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa

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6.2.2 | Mobile phase containers

6.2.3. Volumetric glassware as required by standard and sample preparations

6.2.4 Tips for adjustable automatic pipets

6.2.5 10mL plastic luer lock syringe

6.2.6 0.2 or 0.45um nylon syringe filters

6.2.7. Microcentrifuge tubes

6.2.8 Weigh paper or weigh boats

6.3 Equipment
6.3.1 Suitable oralictt 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. Vortex

6.3.4 Stir Plate

6.3.5 Centrifuge
6.3.6 Wrist action shaker

6.3.7. Adjustable automatic pipets as required by standard and sample preparations

6.4 Mobile Phase and Buffer Preparation

6.4.1 Mobile Phase A (40mM NaH2PO, in H20, pH= 7.8)- is prepared by dissolving

4.782 NaH2POs in 950ml H20, then titrating to pH 7.8 with NaOH solution.
After pH adjustment, bring up to 1 L with H20.

6.4.2 Mobile Phase B (ACN: MeOH: H20 (45:45:10))- is prepared by adding 450mL

ACN, 450mL MeOH and 100mL H20 to a 1L volumetric flask and mix well.

6.4.3 Diluent- H20O.

6.4.4 0.2M Borate buffer- is prepared by dissolving 7.6274g of Na2B4O07. 10H20 into
90ml of H20, titrating to pH= 10.2 with NaOH solution. After pH adjustment,

bring up to 100ml with H20.

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6.5 Stock Standard Preparation

6.5.1 Accurately weigh and transfer about 25 mg of reference standard into a 50-mL

volumetric flask.

6.3.2 Dissolve in and dilute to volume using Diluent.
6.6 Working Standard Preparation

6.6.1 Transfer 5.0 mL of the Stock Standard into a 25-mL volumetric flask.

6.6.2 Dilute to volume using Diluent.

6.6.3 Alternate standard preparations are acceptable provided that the Working

Standard concentration is within the linear range listed below

6.7 Sample Preparation
6.7.1 Specific sample testing details are provided in each products profile. Ifa specific

testing details section is not available, then follow preparation procedure as

described below, maintaining concentration within the linear range listed below.

6.7.2 The linear range of the method for each analyte is listed in the table below. The
concentration of the sample preparation must be within the linear range.

Analyte Linear Range (mcg/mL)
Leucine 25 - 400
Taurine 10 - 600
Theanine 12 — 800
B-alanine, aspartic acid, carnosine,
glutamine, isoleucine, lysine,
10- 100
phenylalanine, threonine, tyrosine,
and valine
6.7.3 For raw materials: weigh no less than 25 mg into a suitably sized volumetric

flask of no less than 50 mL volume to generate an analyte concentration that is
within the validated linearity range. Dilute to 2/3 of the flask volume with

Diluent, shake mechanically for 30 minutes, and dilute to volume using Diluent.

6.7.4 For solid dose finished products: Combine and homogenize no less than ten
dosage units. Based on the label claim and fill weight (for capsules) or tablet

weight per dose, weigh no less than 25 mg of the pooled dosages into a suitably

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sized volumetric flask of no less than 50 mL to generate an analyte concentration

that is within the validated linearity range. Dilute the sample to 2/3 of the flask
volume with Diluent, shake mechanically for 30 minutes, and dilute to volume

using Diluent.

6.7.5 For liquid dose finished products: Use a TC pipet to transfer no less than 2.0 mL
of the product into a suitably sized volumetric flask of no less than 25 mL to

generate an analyte concentration that is within the validated linearity range.

Wipe the outside of the pipet, and rinse the pipet three times with Diluent
collecting the rinses in the volumetric flask. Dilute to volume using Diluent.

6.7.6 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 pooled and homogenized dosages into a glass beaker.

Dissolve the sample in Diluent, transfer the dissolved sample into a suitably
sized volumetric flask of no less than 25 mL volume to generate an analyte

concentration that is within the validated linearity range using small portions of

Diluent to ensure complete transfer, and dilute to volume with Diluent.

6.7.7 Perform additional dilutions as required using Diluent
6.7.8 Filter an aliquot of the sample solution through a 0.45 um membrane discarding

the first 2 - 3 mL of filtrate before collecting a portion in a vial for analysis.

6.7.8.1 Alternatively, the solution may be centrifuged to remove particulates
provided that the final solution is clear.

6.7.9 For samples that have multiple amino acids, care should be made to assure the

concentrations are as low as possible due to the competitive reaction for the

derivatization reagent.

6.8 Test Conditions
6.8.1 Gradient-Isocratic (For Taurine)

Time YA %B

0.00 70 30

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1] 70 30

6.8.2 Gradient-Isocratic (For Leucine)

Time YA %B

0.00 45 55

12 45 2

6.8.3 Gradient-Isocratic (For L-Theanine/ B-alanine/Tyrosine)

Time MA %B

0.00 70 30

16 70 30
6.8.4 Gradient-linear (For Glutamine, Threonine, Valine, Phenylalanine, Isoleucine,

Lysine, Aspartic acid and Carnosine).

Time YA %B

0.00 100 0

1.90 100 0

18.1 43 57.0
18.6 0 100

22.3 0 100

23.2 100 0

26.0 100 0

6.8.5 Column- ZORBAX Eclipse AAA, 3.5u, 120A, 4.6 X 250mm

6.8.6 Flow Rate- 1.0mL/min

6.8.7 Wavelength- 338nm

6.8.8 Column Temperature- 40°C

6.8.9 Recommended 3-D Spectral Range- 200nm to 500nm
6.8.10 Auto-injector program

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6.8.10.1 Taurine, Leucine: injection volume- 5uL

Draw 10.0 uL from vial A(borate buffer), 200 ul/min speed

Draw 0.0 uL from vial B (H20), 200 ul/min speed

Draw 5.0 uL from vial C (amino acid solution), 200 ul/min speed

Mix 15.0 uL in air, max speed, 5 times

Wait 1.00 min

Draw 0.0 uL from vial B (H20), 200 ul/min speed
Draw 30.0 uL from vial D (OPA), 200 ul/min speed

Mix 45.0 uL in air, max speed, 5 times

Inject

Needle wash in vial B (H2O), 1 times

Valve bypass

6.8.10.2 L-Theanine, B-alanine, Tyrosine Glutamine, Threonine, Valine,
Phenylalanine, Isoleucine, Lysine, Aspartic acid and Carnosine:

injection volume- 2.5 uL

Draw 5.0 uL from vial A(borate buffer), 200 ul/min speed

Draw 0.0 uL from vial B (H20), 200 ul/min speed

Draw 2.5 uL from vial C (amino acid solution), 200 ul/min speed

Mix 7.5 uL in air, max speed, 5 times

Wait 0.5 min
Draw 0.0 uL from vial B (H20), 200 ul/min speed

Draw 5.0 uL from vial D (OPA), 200 ul/min speed

Mix 12.5 uL in air, max speed, 6 times

Draw 32 uL from vial B (H20), 200 ul/min speed

Mix 44.5 uL in air, max speed, 8 times

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Inject

Wait 0.5min

Valve bypass

Note: This program is an example; the positions for the reagents can

be changed based on needs.
6.9 Recommended Sequence

6.9.1 Make at least 2 injections of a Blank (Diluent).

6.9.2 Make five injections of the Working Standard.

6.9.3 Make a single injection of each Sample Preparation.

6.9.4 Make a single injection of the Working Standard after every ten samples and at

the end of the run.

6.10 System Suitability
6.10.1 The %RSD of five consecutive injections of the Working Standard is NMT 5.0%.

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

6.11 Column Wash and Storage

6.11.1 Rinse the column with H2O / ACN (90/10) at 1 mL/min for at least 15 min.

6.11.2 Rinse the column with H2O / ACN (50/50) at 1 mL/min for at least 10 min.

6.11.3 Store the column with H2O / ACN (50/50).

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

6.112 .1 9Y % assaayy =— R R x WtSV s o t reg a X P x. V a sEp pi x . a SS a x 100

Ry Sample peak area

R, Mean standard peak area

Wt..qg 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

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SA Sample amount in mg (solids) or mL (liquids)

Vn 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.

LA Label amount in mg per dose or 1 for raw materials

7.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 2 | 03/25/15 | Changed SOP format. Added L-Theanine. | 15-0225 | B. Johns |
| 3 | 03/26/19 | Scheduled review. added new analytes. | 19-0240 | J. Maignan |
| 4 | 03/16/23 | Update for clarity, ease of use, and consistency with current methods CC- and lab practices. | 23-0141 | S. Sassman |