D-770

Apoaequorin Determination by HPLC using UVVIS Spectroscopy

Section D — Laboratory Operations and Specifications Revision 2 24 pages

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JAN LABS Change Control Request Form
Form: C-403-F1 CCR No. CC-22-0477 Revision: 8

CCR No CC-25-0510 Rev 0 Due Date 01/18/25

SECON 1- wang’ oh ae Cnitiator)
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SOP Apoaequorin Determination by HPLC using D-770 7 |
UV-VIS Spectroscopy 7 | | ales

: Change Summary :

Implemented standard recovery requirement

Rationale
Per CAPA-25-0074

+(i.e. Standard Operating Procedure, Forms, Standard Test Methods, Specifications, Product Profiles, Protocols, Reports, Master
Batch Records, Materials, Packaging Components, Labeling, Equipment, Suppliers)
Change Request Approved By/Date: J Ly {2 | | 4 2S

1. Initial Changes made by/Date: AW S (2 N26

2. Additional Changes made by/Date: es

3. Additional Changes made by/Date: oan

SECTION 2 — Change Control Review and Impact Assessment/Requirements for Closure (DC/Reviewer/Approver) —
No. Activity - Responsible Assigned By/Date | Complete

ae _ a

3. L

[SOP

12 Change Control Request Form
J! N LABS Form: C-403-F1 CCRNo. CC-22-0477 Revision: 8

CCR No CC-25-0510 Rev 0 Due Date 01/18/25

Customer Notification Required: LI] Yes M1 No Date of Notification: N/A (attach evidence)

a : SECTION 3 — Approvals [Reviewed/Approved (Y) Yes or (N) No-Initials/Date. If No, provide explanation |
ane First Routing Second Routing
Department rint Name Y/N Initials Date Y/N Initials Date

QA Melisa Maples Y VM Olble-Lhe |6 Z
QA Nicole Motley y “Vy MEL (1-09- 1G oN
a
Ls| Ny,
SET X

ail aa NNNN

— a IN

SECTION 4 — Change Control Implementation (DC)
Document Effective |
Date O\- D§-7b By IAT>O Date &(-~O4-2¢

oe SECTION 5 - Change Control Closure (DC)

AI requirements included in the change control verified as complete B
and closed y

Comments
ee

[SOP

Standard Operating Procedure SOP Number Revision
@®HB Lloanb s Apoaequori. n Determi—na—ti on by EffecDt-i7v7e0 D ate Pp8

| HPLC using UV/VIS Spectroscopy ra
Page 1 of 11
Written by/ Date Reviewed by/ Date Approved by/ Date
AT v 1 | \ Us MM Ol deze PUNC 01 08-26

Title: QC Lab Manager Title: Analytical QA Specialist | Title: Quality Director

1.0 Purpose

The purpose of this procedure is to define the method for the quantitation and/or identification

of Apoaequorin in raw materials and finished product dietary supplements using HPLC and
UV/VIS spectrophotometry.

2.0 Scope

This procedure applies to the quantification and identification of Apoaequorin in raw materials
and finished products in the QC laboratory at Ion Labs. Apoaequorin is a good chromophore and

was measured at 215 nm.

3.0 Responsibility

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

3.2 It is the responsibility of QC Laboratory Management 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 UV/VIS — Ultraviolet and visible electromagnetic spectrum

4.2 Tris — Tris(hydroxymethyl)aminomethane base

4.3 H3POs — Phosphoric Acid

4.4 EDTA —Ethylenediaminetetraacetic acid disodium salt dihydrate

4.5. NaCl — Sodium chloride

4.6 TFA — Triflouroacetic acid

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 2 of 11
Spectroscopy
4.7 ACN — Acetonitrile

4.8 CofA — Certificate of analysis

4.9 HeO- Water (> 18.2 MQ-cm)

4.10 Apoaequorin — Fluorescent protein isolated from jellyfish

5.0 References

5.1 MV-LAB-18-167, Protocol, Apoaequorin Determination using HPLC with UV/VIS
Spectroscopy

5.2 PRTCL-24-0033, Protocol, Supplemental Validation of D-770 for Determination of

Apoaequorin in Chewable Gels by HPLC-UV

6.0 Supplies

6.1 Chemicals: All reagents are ACS grade or better.

6.1.1 H2O

6.1.2 ACN

6.1.3 TFA

6.1.4 Tris

6.1.5 Apoaequorin reference standard

6.1.6 NaCl

6.1.7 H3PO4

6.1.8 EDTA

6.2 Glassware

6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa

6.2.2 HPLC vial inserts

6.2.3 Erlenmeyer Flasks

6.2.4 Mobile Phase Containers

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 § 3 of 11
Spectroscopy
6.2.5 Volumetric Flasks

6.2.6 Volumetric Pipets

6.3 Disposables

6.3.1 Pipette Tips

6.3.2 1.5mL microfuge tubes

6.3.3 Razor Blades

6.3.4 Disposable Plastic Luer Lock Syringe

6.3.5 0.45um PVDF low-binding protein filters or equivalent or PTFE

6.3.6 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 Micro Analytical Balance

6.4.4 Stir Plate

6.4.5 Wrist-action Shaker

6.4.6 Microfuge

6.4.7 Adjustable Pipettes

7.0 Preparation of Mobile Phase, Dissolution Buffer, Samples, and Standards

7.1 Mobile Phase Preparation

7.1.1 All mobile phases should be prepared in glass only.

7.1.2 Mobile Phase A — 30/70 ACN/H20 + 0.1% TFA

7.1.2.1 Transfer 300mL ACN to a 1000-mL glass bottle.

7.1.2.2 Add 700mL H20.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 8 4 of 11
Spectroscopy
7.1.2.3. Carefully add 1.0mL TFA, and mix well.

7.1.3. Mobile Phase B— ACN + 0.1% TFA

7.1.3.1 Transfer 1000mL ACN to a 1000-mL glass bottle.

7.1.3.2 Carefully add 1.0mL TFA, and mix well.

7.1.4 Diluent — 60/40 ACN/H20

7.1.4.1 Transfer 600mL ACN to a 1000-mL glass bottle.

7.1.4.2 Add 400mL H20, and mix well.

7.1.5 Resuspension Buffer —50mM TRIS, 25mM NaCl, IlmM EDTA, pH8.5

7.1.5.1 Transfer 1.46 g of NaCl to a 1000-mL glass bottle.

7.1.5.2 Add 0.372 g of EDTA.

7.1.5.3. Add 6.06 g of TRIS.

7.1.5.4 Add 1000mL of H20, and mix until dissolved.

7.1.5.5 Adjust to pH 8.5 using H3PQO..

7.1.6 Standard Preparation

7.1.6.1 The linear range of the method is 0.0068 — 0.8 mg/mL. All final standard

and sample preparations must be within this range. Use the actual purity

from the CofA or the standard certification for apoaequorin reference
material for calculations.

7.1.6.2 All standards are prepared in duplicate (Std A and Std B).

7.1.6.3 Mix solutions gently, being sure to not allow foam to form. Do NOT

vortex. Do NOT re-freeze the reference standard after use. Allow to
warm to room temperature before use.

7.1.6.3.1 Examine the 8.3mg/ml stock standard after thawing from

frozen state. Mix Standard solution gently before making
dilution for the working standard.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 8 5 of 11
Spectroscopy
Note: The Standard Material can precipitate out of
solution causing the standard concentration to be lower
than the expected 8.3mg/ml. The precipitation of the
standard from the solution causes high results for assay
testing. It is essential to verify standard is fully

dissolved before proceeding with dilutions.

Examine the 8.3mg/ml stock standard after thawing from frozen state.
Mix Standard solution gently before making dilution for the working

standard.
7.1.6.4 The standard is prepared by using glass volumetric pipets and glass

volumetric flasks.

7.1.6.5 The target concentration of the working standard is generally 0.5
mg/mL, however, see 7.1.6.4.1 if analyzing gummies. The working

standard is prepared in Diluent from a pre-prepared stock. Choose

aliquot and final volumes to result in a final concentration of about 0.5
mg/mL.

Example: Prepare a 0.5mg/mL working standard from an 8.3mg/mL

stock. Using an aliquot volume of 1.0mL, first calculate the
final volume.

Mi*Vi=MoV*2

8.3 mg/mL (stock) * ImL=0.5mg/mL * V2 mL

V2=16.6mL (most convenient final volume is 16mL)

Using a glass volumetric pipet, add 1.0mL of 8.3mg/mL
stock to a 25-mL glass volumetric flask. Using a glass

volumetric pipet, add 15.0mL of Diluent to the same 25-
mL volumetric flask. Mix gently. Do not dilute to volume.

Std Conc = 8.3mg/mL * ImL / 16mL = 0.51875 mg/mL

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 8 6 of 11
Spectroscopy
7.1.6.5.1 If analyzing gummies, perform an additional 1:25 dilution
in diluent using volumetric glassware for a working
standard concentration of ~20ug/ml.

7.1.6.6 Pass standard through 0.45um PVDF low-protein-binding filter (or
equivalent), being sure to discard the first few milliliters to waste.

7.1.6.7 Add standard to a2mL HPLC vial. (A low-volume insert can be used to

extend working life of the 0.5mg/mL working standard.)

7.1.6.8 The 0.5mg/mL working standard can be stored for up to 8 weeks at 2-

8°C. (Refrigerated storage does not apply to the 20ug/ml working
standard, this standard must be prepared and used fresh.)

7.2 Sample Preparation

7.2.1 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 above.

7.2.2 The target concentration of the final sample solution is generally 0.5 mg/mL.
Gummies are analyzed at ~20ug/mL. Sample preparation examples are provided

below in 7.2.6. The final sample concentration must be within the linear range of
the method.

For raw materials, consult the potency listed on the vendor COA for calculation
7.2.3 of the required sample weight.

7.2.4 For finished products, a composite of no less than 10 dosage units is generally

used as the sample for analysis.

7.2.5 The sample stock solution should be prepared in Resuspension Buffer. Carefully
and gently mix to ensure powder is fully suspended. Then, stir or mix on a wrist

action shaker at low speed for at least 1 hour being careful to not allow foam to

form. For stirring, dilute to volume first then add a stir bar prior to stirring for at
least 1 hour. For shaking, dilute to 2/3 volume, shake for at least 1 hour, then

dilute to the final volume and mix gently.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 8 7 of 11
Spectroscopy
7.2.6 A 10x dilution of the sample stock solution should be performed with Diluent to
dilute the Resuspension Buffer salts. Filter through a 0.45um PVDF low-binding

protein filter (or equivalent) being sure to discard the first few milliliters before
collecting a portion in a HPLC vial for analysis.

Example: Prevagen capsule (label claim = 20 mg, dosage unit = 467 mg)

Prepare 10mL of a 0.5mg/mL solution

Combine the fill material from 20 capsules. Prepare a sample stock
solution by transferring 5.8g of the combined fill material to a 50mL

volumetric flask, and gently dissolving in Resuspension Buffer.
Transfer 1.0mL of the stock solution to a 10-mL volumetric flask, and

dilute to volume using Diluent.

20mg / 0.467g * 5.8 g/50mL * ImL/ 10mL = 0.4968 mg/mL

Example: Apoaequorin raw material (given 100% potency)

Prepare 10mL of a 0.5mg/mL solution

Prepare a sample stock solution by transferring 250mg of the

combined fill material to a 50mL volumetric flask, and gently
dissolving in Resuspension Buffer. Transfer 1.0mL of the stock

solution to a 10-mL volumetric flask, and dilute to volume using

Diluent.

250mg /50mL * lmL / 10mL = 0.5 mg/mL

Example: Prevagen gummy (label claim = 5 mg, dosage unit = 1 gummy)

Prepare 100mL of a 0.2mg/mL solution

Quarter four gummies with a razor and prepare a sample stock solution
by transferring to a 125mL Erlenmeyer flask and diluting to ~80mL

with Resuspension Buffer. Parafilm and stir gently on a stir plate for

1 hour. Ensure gummies are completely dissolved. Quantitatively
transfer to a 100mL volumetric flask, QS to volume and gently mix.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 8 8 of 11
Spectroscopy
Transfer 5.0mL of the stock solution to a 50-mL volumetric flask, and
dilute to volume using Diluent.

20mg /100mL * 5mL /50mL = 20 ug/mL

8.0 Test Conditions

8.1 Gradient

Time YA %B

0.00 90 10

0.3 90 10

2.7 50 50

2.73 10 90

3.57 10 90

3.6 90 10

6.0 90 10

8.2 Column — Phenomenex Jupiter, C4, Sum, 300A, LC column, 150mm x 4.6mm, or

equivalent.

8.3 Flow Rate — 2.0mL/min

8.4 UV Detection —215nm

8.5 Recommended 3D Spectral Range — 200nm — 300nm

8.6 Injection Volume — 15.5uL

8.7. Column Temperature — 40°C

8.8 Retention Time — about 2.7 minutes

8.9 Recommended Sequence

8.9.1 Make at least two injections of the diluent.

8.9.2 Make five injections of Standard Solution A.

[SOP

> tandard Opperearteinnng e Procedure SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 8 9 of 11
Spectroscopy

8.9.3 Make two injections of Standard Solution B.

8.9.4 Make a single injection of each Sample Preparation.

8.9.5 Makea single injection of Standard Solution A after every six samples and at the
end of the run.

8.10 System Suitability Requirements

8.10.1 The %RSD of the first five standard injections is NMT 5.0%.

8.10.2 The % recovery of Working Standard A, using Working Standard B is 98-102%

8.10.3 The %RSD of all Standard Solution A injections is NMT 5%.

8.11 Column Wash and Storage

8.11.1 Wash the column with ACN/H20 (50/50) at 1 mL/min for at least 15 min.

8.11.2 Store the column with ACN/H20 (50/50).

9.0 Calculations

= Adstd ¥ Vspl y SS
9.1 % assay = > X Cota X Teta X= Xi X 100

Ry Sample peak area

R, Mean standard peak area

C.,q Concentration of pre-prepared Stock Standard in mg/mL

Adstq Aliquot of Stock Standard used to prepare Working Standard in mL

Vstq Final Volume of the Working Standard in mL

SA Sample amount in mg (solids) or mL (liquids), or 4 for gummies

Vsp1 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 and gummies.

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

[SOP

Standard Operating Procedure Snr Ne —_ _
Apoaequorin Determination by HPLC using UV/VIS D-770 8 10 set
Spectroscopy

9.2. % Difference from CofA

| Ap - Acofa
% Difference from CofA = Acota x 100

Ap _ Assay value determined

Acofa Assay value reported on manufacturer’s Certificate of Analysis

10.0 Example Chromatography

10.1 Blank

Blank
1100
4000-7
900-
800-
700-
sty 600-
< 500-
400-
300-
200-
1000- VY /\\
-100-1
02040608 112141618 2 22242628 3 32343638 4 42444648 5 52545658 6
Fime [min]
rking Standard
23AS090
1100
1000-
900-
800-
700-
600-
< 500-
S 400-
300-
200-
100-
-100- Ls & Lj Lj zi Ly £ LJ Lj 3 cy = & = Eg r ¥ t = . a ¥ ¥ Ly + b 3 Ey z 3 Ey =
0.2040608 1 12141618 2 222426 28 3 3234363.8 4 42444648 5 52545658 6
Time [min]
236
nirouqeaopA
979.2 10.2. Sample

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 11 of 11
Spectroscopy
230417
1100
10007
900-
800-
7007
600-
< 500-
400-
300-
200-
100-
—“\
-100-1
nirouqeaopA
_/\\—
02040608 1 412141618 2 22242628 3 32343638 4 42444648 5 52545658 6
Time [min]
11.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/02/19 | New. N/A J. Maignan Update to reflect current practices and for clarity, add reference to | - | - |
| 1 | 04/15/22 | the validation, add details for prep of suspension buffer, add Cc- recommended sequence, add system suitability requirements, add column wash and storage, add example chromatography. | 22-0177 | S. Sassman |
| 2 | 11/16/22 | Clarified spectral range. Remove retention time requirement. CC- Add instruction to check the product profile for test details, remove | 22-0441 | J. Sassman |
| 3 | 02/23/23 | language requiring in-process validation for new products, remove CC- spectral match as system suitability requirement. | 23-0095 | S. Sassman |
| 4 | 12/09/23 | Added more representative chromatography. CC- | 23-0592 | J. Sassman |
| 5 | 04/19/24 | Revise method to include expanded linear range and new finished CC- product matrix. | 24-0165 | C. Perry |
| 6 | 11/27/24 | Added calculation for determination of % difference from value on CC- manufacturer’s Certificate of Analysis | 24-0538 | K. Bittler |
| 7 | 04/30/25 | Added standard solution examination after thawing CC- | 25-0196 | _ K. Bittler |
| 8 | 12/18/25 | Implemented standard recovery requirement. CC- | 25-0510 | A. Shannon |

[SOP

Standard Operating Procedure SOP Number Revision
&A@) Lap) lLoanb s Apoaequori. n Determianati on by EffecDt-i7v7e0 D ate Pa87ge

HPLC using UV/VIS Spectroscopy Page 1 of 11

Written by/ Date Reviewed by/ Date Approved by/ Date

Title: C9h€emistQC Lab Title: Heeulioe Ades Title: QA/OC Quality Director
Manager SuperviserAnalytical OA
Specialist

The purpose of this procedure is to define the method for the quantitation and/or identification

of Apoaequorin in raw materials and finished product dietary supplements using HPLC and

UV/VIS spectrophotometry.

2.0 Scope

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

and finished products in the QC laboratory at Ion Labs. Apoaequorin is a good chromophore and
was measured at 215 nm.

3.0 Responsibility

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

3.2 It is the responsibility of QC Laboratory Management 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 UV/VIS — Ultraviolet and visible electromagnetic spectrum

4.2 Tris — Tris(hydroxymethyl)aminomethane base

4.3 H3PO« — Phosphoric Acid

4.4 EDTA — Ethylenediaminetetraacetic acid disodium salt dihydrate

aS NaCl — Sodium chloride

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 2 of 11
Spectroscopy
4.6 TFA — Triflouroacetic acid

4.7 ACN — Acetonitrile

4.8 CofA — Certificate of analysis

4.9 H20 — Water (> 18.2 MQ-cm)

4.10 Apoaequorin — Fluorescent protein isolated from jellyfish

5.0 References

5.1 MV-LAB-18-167, Protocol, Apoaequorin Determination using HPLC with UV/VIS
Spectroscopy

5.2 PRTCL-24-0033, Protocol, Supplemental Validation of D-770 for Determination of

Apoaequorin in Chewable Gels by HPLC-UV

6.0 Supplies

6.1 Chemicals: All reagents are ACS grade or better.

6.1.1 H20

6.1.2 ACN

6.1.3 TFA

6.1.4 Tris

6.1.5 Apoaequorin reference standard

6.1.6 NaCl

6.1.7 H3PO4

6.1.8 EDTA

6.2 Glassware

6.2.1. HPLC vials, 12mm x 32mm with screw cap enclosures with septa

6.2.2. HPLC vial inserts

6.2.3. Erlenmeyer Flasks

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 3 of 11
Spectroscopy
6.2.4 Mobile Phase Containers

6.2.5 Volumetric Flasks

6.2.6 Volumetric Pipets

6.3 Disposables

6.3.1 Pipette Tips

6.3.2 1.5mL microfuge tubes

6.3.3 Razor Blades

6.3.4 Disposable Plastic Luer Lock Syringe

6.3.5 0.45um PVDF low-binding protein filters or equivalent or PTFE

6.3.6 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. Micro Analytical Balance

6.4.4 Stir Plate

6.4.5 Wrist-action Shaker

6.4.6 Microfuge

6.4.7 Adjustable Pipettes

7.0 Preparation of Mobile Phase, Dissolution Buffer, Samples, and Standards

7.1 Mobile Phase Preparation

7.1.1. All mobile phases should be prepared in glass only.

7.1.2. Mobile Phase A — 30/70 ACN/H20 + 0.1% TEA

7.1.2.1. Transfer 300mL ACN to a 1000-mL glass bottle.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 4 of 11
Spectroscopy
7.1.2.2 Add 700mL H20.

7.1.2.3 Carefully add 1.0mL TFA, and mix well.

7.1.3 Mobile Phase B— ACN + 0.1% TFA

7.1.3.1 Transfer 1000mL ACN to a 1000-mL glass bottle.

7.1.3.2 Carefully add 1.0mL TFA, and mix well.

7.1.4 Diluent — 60/40 ACN/H20

7.1.4.1 Transfer 600mL ACN to a 1000-mL glass bottle.

7.1.4.2 Add 400mL H20, and mix well.

7.1.5 Resuspension Buffer —-50mM TRIS, 25mM NaCl, lmM EDTA, pH8.5

7.1.5.1 Transfer 1.46 g of NaCl to a 1000-mL glass bottle.

7.1.5.2 Add 0.372 g of EDTA.

7.1.5.3 Add 6.06 g of TRIS.

7.1.5.4 Add 1000mL of H20, and mix until dissolved.

7.1.5.5 Adjust to pH 8.5 using H3POs.

7.1.6 Standard Preparation

7.1.6.1 _The linear range of the method is 0.0068 — 0.8 mg/mL. All final standard

and sample preparations must be within this range. Use the actual purity

from the CofA or the standard certification for apoaequorin reference
material for calculations.

F71617.1.6.2 All standards are prepared in duplicate (Std A and Std B).

71627.1.6.3 Mix solutions gently, being sure to not allow foam to form. Do

NOT vortex. Do NOT re-freeze the reference standard after use. Allow

to warm to room temperature before use.

FA6217.1.6.3.1 Examine the 8.3mg/ml stock standard after thawing
from frozen state. Mix Standard solution gently before
making dilution for the working standard.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 S of 11
Spectroscopy
Note: The Standard Material can precipitate out of
solution causing the standard concentration to be lower
than the expected 8.3mg/ml. The precipitation of the
standard from the solution causes high results for assay

testing. It is essential to verify standard is fully
dissolved before proceeding with dilutions.

Examine the 8.3mg/ml stock standard after thawing from frozen state.
Mix Standard solution gently before making dilution for the working

standard.
716:37.1.6.4 The standard is prepared by using glass volumetric pipets and glass

volumetric flasks.

716-47.1.6.5 The target concentration of the working standard is generally 0.5
mg/mL, however, see 7.1.6.4.1 if analyzing gummies. The working

standard is prepared in Diluent from a pre-prepared stock. Choose

aliquot and final volumes to result in a final concentration of about 0.5
mg/mL.

Example: Prepare a 0.5mg/mL working standard from an 8.3mg/mL

stock. Using an aliquot volume of 1.0mL, first calculate the

final volume.

Mi*Vi=Mo2V*2

8.3 mg/mL (stock) * ImL=0.5mg/mL * V2 mL

V2=16.6mL (most convenient final volume is 16mL)

Using a glass volumetric pipet, add 1.0mL of 8.3mg/mL
stock to a 25-mL glass volumetric flask. Using a glass

volumetric pipet, add 15.0mL of Diluent to the same 25-

mL volumetric flask. Mix gently. Do not dilute to volume.

Std Conc = 8.3mg/mL * ImL / 16mL = 0.51875 mg/mL

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 6 of 11
Spectroscopy
716447.1.6.5.1 If analyzing gummies, perform an additional 1:25
dilution in diluent using volumetric glassware for a
working standard concentration of ~20ug/ml.

71657.1.6.6 Pass standard through 0.45um PVDF low-protein-binding filter
(or equivalent), being sure to discard the first few milliliters to waste.

71.6-67.1.6.7 Add standard to a 2mL HPLC vial. (A low-volume insert can be

used to extend working life of the 0.5mg/mL working standard.)

F16-77.1.6.8 The 0.5mg/mL working standard can be stored for up to 8 weeks

at 2-8°C. (Refrigerated storage does not apply to the 20ug/ml working
standard, this standard must be prepared and used fresh.)

me: Sample Preparation

7.2.1 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 above.

7.2.2 The target concentration of the final sample solution 1s generally 0.5 mg/mL.
Gummies are analyzed at ~20ug/mL. Sample preparation examples are provided

below in 7.2.6. The final sample concentration must be within the linear range of
the method.

7.2.3 For raw materials, consult the potency listed on the vendor COA for calculation
of the required sample weight.

7.2.4 For finished products, a composite of no less than 10 dosage units is generally

used as the sample for analysis.

7.2.5 The sample stock solution should be prepared in Resuspension Buffer. Carefully
and gently mix to ensure powder is fully suspended. Then, stir or mix on a wrist

action shaker at low speed for at least 1 hour being careful to not allow foam to

form. For stirring, dilute to volume first then add a stir bar prior to stirring for at
least 1 hour. For shaking, dilute to 2/3 volume, shake for at least 1 hour, then

dilute to the final volume and mix gently.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 7 of 11
Spectroscopy
7.2.6 A 10x dilution of the sample stock solution should be performed with Diluent to
dilute the Resuspension Buffer salts. Filter through a 0.45um PVDF low-binding

protein filter (or equivalent) being sure to discard the first few milliliters before

collecting a portion in a HPLC vial for analysis.

Example: Prevagen capsule (label claim = 20 mg, dosage unit = 467 mg)

Prepare 10mL of a 0.5mg/mL solution

Combine the fill material from 20 capsules. Prepare a sample stock
solution by transferring 5.8g of the combined fill material to a 50mL

volumetric flask, and gently dissolving in Resuspension Buffer.

Transfer 1.0mL of the stock solution to a 10-mL volumetric flask, and
dilute to volume using Diluent.

20mg / 0.467g * 5.8 g/50mL * ImL/ 10mL = 0.4968 mg/mL

Example: Apoaequorin raw material (given 100% potency)

Prepare 10mL of a 0.5mg/mL solution

Prepare a sample stock solution by transferring 250mg of the

combined fill material to a 5OmL volumetric flask, and gently

dissolving in Resuspension Buffer. Transfer 1.0mL of the stock
solution to a 10-mL volumetric flask, and dilute to volume using

Diluent.

250mg /50mL * ImL/ 10mL = 0.5 mg/mL

Example: Prevagen gummy (label claim = 5 mg, dosage unit = 1 gummy)

Prepare 100mL of a 0.2mg/mL solution

Quarter four gummies with a razor and prepare a sample stock solution

by transferring to a 125mL Erlenmeyer flask and diluting to ~80mL
with Resuspension Buffer. Parafilm and stir gently on a stir plate for

1 hour. Ensure gummies are completely dissolved. Quantitatively
transfer to a 100mL volumetric flask, QS to volume and gently mix.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 8 of 11
Spectroscopy
Transfer 5.0mL of the stock solution to a 50-mL volumetric flask, and
dilute to volume using Diluent.

20mg /100mL * 5mL /50mL = 20 ug/mL

8.0 Test Conditions

8.1 Gradient

Time YoA %B

0.00 90 10

0.3 90 10

re | 50 50

2.73 10 90

3.57 10 90

3.6 90 10

6.0 90 10

8.2 Column — Phenomenex Jupiter, C4, 5um, 300A, LC column, 150mm x 4.6mm, or

equivalent.

8.3 Flow Rate — 2.0mL/min

8.4 UV Detection —-215nm

8.5 Recommended 3D Spectral Range — 200nm — 300nm

8.6 Injection Volume — 15.5uL

8.7 Column Temperature — 40°C

8.8 Retention Time — about 2.7 minutes

8.9 Recommended Sequence

8.9.1 Make at least two injections of the diluent.

8.9.2 _Make five injections of Standard SolutionA.

[SOP

Standard Operating Procedure
P e SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS D-770 97 9 of 11
Spectroscopy

$9.28 .9.3 Make two injections of Standard Solution B.

$-938.9.4 Make a single injection of each Sample Preparation.

$-9-48.9.5 Make a single injection of the Standard Solution_A after every six samples
and at the end of the run.

8.10 System Suitability Requirements

8.10.1 The %RSD of the first five standard injections is NMT 5.0%.

$1018.10.2 The % recovery of Working Standard A, using Working Standard B is 98-

102%

81028.10.3 The %RSD of all Sstandard Solution A injections is NMT 5%.

8.11 Column Wash and Storage

8.11.1 Wash the column with ACN/H20 (50/50) at 1 mL/min for at least 15 min.

8.11.2 Store the column with ACN/H20 (50/50).

9.0 Calculations

Ry Sample peak area

R, Mean standard peak area

Csq¢ Concentration of pre-prepared Stock Standard in mg/mL

Adstq Aliquot of Stock Standard used to prepare Working Standard in mL

Vezq Final Volume of the Working Standard in mL

SA Sample amount in mg (solids) or mL (liquids), or 4 for gummies

Vsp1 | 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 and gummies.

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 SH 10 of 11
Spectroscopy
LA Label amount in mg per dose or 1 for raw materials

9.2. % Difference from CofA

| Ap - AcofA
% Difference from CofA = 100
AcofA
Ap Assay value determined

Acota Assay value reported on manufacturer’s Certificate of Analysis

10.0 Example Chromatography

10.1. Blank

Blank
1100
10007
900-
800
700-
600-
< 500-
400-
300-
2007
100- US f/f
-100- 02040608 112141618 222242628 3 32343638 4 42444648 5 52545658 6
Time [min]
rking Standard
23AS090
1100
1000-
900-
800-
700-
_ 600-
<< 500-7
- 400-
300-
2007
100-
236
nirouqeaopA
979.2 O aN oe J/\—

-100- 02040608 112141618 2 22242628 3 32343638 442444648 5 52545658 6
Time [min]
10.2. Sample

[SOP

Standard Operating Procedure
SOP No Rev Page
Apoaequorin Determination by HPLC using UV/VIS
D-770 87 11 of 11
Spectroscopy
230417
1100
1000-
900-
800-
700-7
600-
—_
< 500-
400-
300-
200-7
100-
——“\
-1007
302.2 nirouqeaopA

_/\—
02040608 1 1412141618 2 22242628 3 32343638 4 42444648 5 52545658 6
Time fmin]
11.0 Revision History
Revision Date Description of Changes CCR # By
0 01/02/19 | New. N/A J. Maignan
Update to reflect current practices and for clarity, add reference to
1 04/15/22 the validation, add details for prep of suspension buffer, add Cc-22-0177 |S. Sassman
recommended sequence, add system suitability requirements, add
column wash and storage, add example chromatography.
2 11/16/22 | Clarified spectral range. Remove retention time requirement. CC-22-0441 | J. Sassman
Add instruction to check the product profile for test details, remove
3 02/23/23 | language requiring in-process validation for new products, remove CC-23-0095 | S. Sassman
spectral match as system suitability requirement.
4 12/09/23 | Added more representative chromatography. CC-23-0592 | J. Sassman
5 04/19/24 Revise method to include expanded linear range and new finished CC-24-0165 C. Perry
product matrix.
6 11/27/24 Added eateulation for determination of % difference from value on CC-24-0538 K. Bittler
manufacturer’s Certificate of Analysis amine
7 04/30/25 | Added standard solution examination after thawing CC-25-0196 K. Bittler
8 12/18/25 | Implemented standard recovery requirement. CC-25-0510 | A. Shannon