D-756

Rosmarinic Acid Determination by HPLC using UV-Vis Spectroscopy

Section D — Laboratory Operations and Specifications Revision 1 8 pages

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1.0 Purpose

The purpose of this procedure is to define the method for the quantitation and/or identification
of rosmarinic acid 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 rosmarinic acid in raw materials

and finished products. Rosmarinic acid is a good chromophore and was measured at 327,
however to minimize any interference, other wavelengths can be used.

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 and Analytical Development Management to

keep this procedure aligned with current practices.

4.0 Definitions

4.] HPLC — High Performance Liquid Chromatography

4.2 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums

4.3 ACN — Acetonitrile

4.4 CofA -— Certificate of Analysis

45 HeO- Water © 18.2 MQ-cm)

4.6 Chlorogenic Acid — 3CQA; 3-O-Caffeoylquinic acid

[SOP

Standard Operating Procedure SOP No | Rev
Rosmarinic Acid Determination by HPLC using D-756 1 . age
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UV/VIS Spectroscopy

5.0 References

5.1 TN-1134, Chlorogenic Acids from Green Coffee by HPLC, Z. Ageel, D. Truong, J.

Preston, S. Lazzaro, and 8. Baugh. Joint Phenomenex and Chromadex

5.2. D-741, SOP, Chlorogenic Acid Determination by HPLC using UV/Vis Spectroscopy

6.0 Supplies

6.1 Chemicals: All reagents are HPLC grade or better.

6.1.1 H20

6.1.2 ACN

6.1.3 Formic acid

6.1.4 Methanol

6.1.5 Chlorogenic acid reference standard

6.2 Glassware

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 1mL Pipette Tips

6.3.3 200uL Pipette Tips

6.3.4 1.5mL microfuge tubes

6.3.5 16mL Test Tubes

6.3.6 Disposable Plastic Luer Lock Syringe — 3mL, 6mL, or 10mL

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Standard Operating Procedure SOP No | Rev
Rosmarinic Acid Determination by HPLC using D-756 I : ae

UV/VIS Spectroscopy

6.3.7 Nylon Syringe Filters, 0.2m

6.3.8 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 Ultrasonic bath

6.4.4 Vortex

6.4.5 Stir Plate

6.4.6 Eppendorf Centrifuge

6.4.7 10mL Pipette

6.4.8 ImL Pipette

6.4.9 200uL Pipette

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

7.1 Mobile Phase A — 0.1% Formic acid in 25% ACN (aq)

Prepared by mixing 250ml ACN, 750ml H20 and Iml of formic acid together

7.2. Mobile Phase B — 0.1% Formic acid in 10% ACN (aq)

Prepared by mixing 100ml ACN, 900ml H20 and I ml of formic acid together

7.3 Dissolution Buffer-60% Methanol (aq)

Prepared by mixing 600ml Methanol and 400ml of H20 together

7.4 Standard Preparation

7.4.1 The linear range of the method is 0.01 mg/mL — 0.10 mg/mL. All standard and
sample preparations must be within the linear range.

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Standard Operating Procedure SOP No | Rev a
Rosmarinic Acid Determination by HPLC using D-756 1 , age
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UV/VIS Spectroscopy

7.4.2 Use the actual purity from the CofA or the standard certification for Rosmarinic
Acid 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 bringing up to the final volume in an appropriate
volumetric flask using dissolution buffer then sonicating for 10 minutes.

7.4.4 Dilutions are prepared using dissolution buffer. 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.

7.5. Sample Preparation

12a For finished products, 10 or more dosage units can be pooled and ground by
mortar and pestle.

7.5.2 Based on the fill weight, tablet weight, or raw material potency, transfer an
amount that is no less than the minimum weight of the analytical balance into a
volumetric flask that is no less than 50 mL to generate an analyte concentration

within the linear range of the method. If necessary, a sample stock solution can

be prepared at higher concentration and diluted using volumetric glassware or
pipets.

7.5.3 The sample is suspended in the final volume and put in the sonicator bath for 10
minutes.

7.5.4 Before injection, insoluble matter should be removed via filtration using a 0.45um

nylon syringe filter. Discard at least 0.5mL of the filtrate before collecting a
portion in an HPLC vial for analysis.

7.5.4.1 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
Rosmarinic Acid Determination by HPLC using D-756 I : aes
UV/VIS Spectroscopy

7.5.5 For raw materials or finished products 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

Time YMA %B Gradient Type

0.00 100 0 0

9.00 100 0 0

9.10 0 100 0

15.00 0 100 0

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

8.3 Flow Rate — 1.0mL/min

8.4 UV Detection — 327 nm

8.5 3D Spectral Range — 220 nm — 400 nm

8.6 Injection Volume - 20uL

8.7 Column Temperature — 30°C

8.8 Retention Time — about 6.5 min

8.9 Recommended Sequence

8.9.1 Make at least 2 injections of the diluent.

8.9.2 Make five (5) injections of Standard Solution.

8.9.3 Make a single injection of each Sample Preparation.

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

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Standard Operating Procedure SOP No | Rev
Rosmarinic Acid Determination by HPLC using D-756 I >
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UV/VIS Spectroscopy

8.10 System Suitability Requirements

8.10.1 The %RSD of five (5) injections of the Working Standard is NMT 5.0%.

8.10.2 The %RSD of all injections of the Working Standard is NMT 5%.

8.10.3 Spectral match over the range 220 nm — 400 nm is NLT 900.

8.10.4 The retention time of the sample is within 0.3 min of the standard.

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

0 as. Bang Wietaet UEspl 88
8.11.1 % assay= si x Va preemie: 100

Ra Sample peak area

R, Mean standard peak area

Wtstg Weight of reference standard in mg

Vieng 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)

Vso 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

[SOP

Standard Operating Procedure SOP No | Rev
Rosmarinic Acid Determination by HPLC using D-756 J nee
7 of8
UV/VIS Spectroscopy

8.12 Example Chromatography

8.12.1 Blank

2 18
E& 1:
0.54 0- i.

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44
“1.55 ' a ? ' i LI LJ i ' LJ ' '
Ae ee ae eG FEB one eos Gia a Agee id ee AL ee ae
Time [min]
8.12.2 Working Standard
DAD1A,Sig=327.0,4.0 Ref-off
50- 3
45; 9
40; 5
354 o
- 30- cO*/1 =
< 26
204
15> to LO
1 eees<a8)k
onao
1 a: 4 5 6 7 8 9 10 1 «2912 13 14 15
Time [min]
8.12.3 Sample

DAD1A,Sig=327.0,4.0 Ref=off
60- 3
La)
2
50- f=
$4”)
2
ae oe
q
< 30-
207 2
Toa te)
0 Noe ee ane
1 2 eae. eee Fo o8 eee Meee | ee eee ee) Coe [
Time [min]

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Standard Operating Procedure SOP No | Rev 7
Rosmarine iec AciLd d Determien ati9 on by HPLC usi6 ng D-756 | aae
8 of 8
UV/VIS Spectroscopy

9.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 08/09/18 | New procedure. N/A J. Maignan Update for consistency with current methods and lab practices, add ] 04/11/22 recommended sequence, add system suitability section, add example CC-22-0173 S. Sassman chromatography, remove extraneous information. | - | - |