D-738
Choline Determination by HPLC using UV-Vis Spectroscopy
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1.0 Purpose
The purpose of this procedure is to define a method for the quantitative analysis and/or
identification of choline in finished products and raw materials using HPLC and UV/VIS
spectrophotometry.
2.0 Scope
This procedure applies to choline quantification and identification. Some excipients and dietary
ingredients used in the finished products can interfere with the analysis of choline. Alternate
wavelengths can be used with justification if interferences are present. Many botanical extracts
can have significant quantities of natural choline.
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 the procedure is being followed.
3.3. It is the responsibility of the QC Laboratory Management and Analytical Development
to keep this procedure current with latest Ion Labs practices.
4.0 Definitions
4.1 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums
4.2 ACN — Acetonitrile
4.3. Na2xHPOs- Sodium Phosphate Dibasic
4.4 H3PO4- Phosphoric Acid
4.5 M20 — Water
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46 QC-— Quality Control
5.0 References
5.1 MV-LAB-13-053, Protocol, L-Choline Determination and Identification by HPLC
5.2 D-793, SOP, Cryogenic Grinding of Chewable Gels
6.0 Reagents, Supplies, Glassware and Equipment
6.1 Reagents: all reagents are HPLC grade or better unless otherwise noted.
6.1.1 H2O
6.1.2 ACN
6.1.3 H3PO4
6.1.4 Choline, Choline Bitartrate, or Choline Chloride traceable standard
6.1.5 NazHPO,
6.2 Supplies and Glassware
6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa
6.2.2 Mobile phase containers
6.2.3. Volumetric glass ware as required by standard and sample preparations
6.2.4 Pipette tips for adjustable pipettes
6.2.5 Plastic luer-lock syringes and 0.45 um syringe filters
6.2.6 Micro centrifuge tubes
6.2.7 Weigh paper and weigh boats
6.3 Equipment
6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
and UV detector with a chromatographic data handling system
6.3.1 Analytical Balance
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Spectroscopy
6.3.2 Stir Plate
6.3.3. Wrist Action Shaker
6.3.4 Vortex
6.3.5 Sonicator Bath
6.3.6 Adjustable Pipettes
7.0 Preparation of Mobile Phase, Diluent, Samples and Standards
7.1 Mobile Phase A - 0.048M Naz HPO, in H2O0
7.1.1. Transfer 8.54 g Na2HPO42H20 (or 6.81 g of anhydrous Na2HPOs) to a 1000-mL
bottle.
7.1.2 Add about 950 mL H20.
7.1.3 Adjust to pH 2.5 with H3PO4.
7.1.4 Transfer to a 1000-mL volumetric flask, and dilute to volume with H20.
7.2 Mobile Phase B — 100% ACN
73 Diluent — Mobile Phase A
7.4 Standard Preparation
7.4.1 The linear range of the method is 0.01 mg/mL — 1.0 mg/mL. The Standard and
Sample Preparations must be within this range.
7.4.2 All standards are prepared by weighing no less than the minimum weight of the
analytical balance then bring up to two thirds their final volumes in an appropriate
volumetric flask using Diluent. Mix on a wrist action shaker for 20 minutes then
inspect to ensure complete dissolution. Sonication for 10 minutes can also be
used to facilitate dissolution. Once the standard is fully dissolved, bring standard
to final volume using Diluent.
7.4.3 To manage large volumes, the standard can be initially prepared at a higher
concentration and further diluted into the linear range using Diluent. Equilibrate
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Spectroscopy 4 i
the solution to room temperature prior performing furthe: dilution.
Dilutions can be made using volumetric glass\ are and/or adjus at e pipettes.
Final dilutions can be prepared in HPLC vials
7.4.4 Working standard concentrations will approxi ne :e the concentration :xpected to
be found in the product being tested based on th: sample dilution iinc calculated
from the label.
7.5 Sample Preparation
7.5.1 Specific sample testing details are provided in ea zh products profil :. . fa specific
testing details section is not available, then fc llow preparation pr cedure as
described below, maintaining concentration w th n the linear range li: ted below
7.5.2 For raw materials: weigh no less than 20 mg in:o 1 suitably sized vc lw aetric flask
of no less than 25 mL volume to generate an ¢ ne yte concentration tk at is within
the validated linearity range. Dilute to volum:« vith Diluent, and 3 »n cate for 10
min.
7.5.3 For solid and liquid dose finished products: ~o nbine and homog er ize no less
than ten dosage units. Based on the label claire ai d fill weight (cap: ul :s), serving
size (powders) or tablet weight per dose, weig11 0 less than 50 mg oi the pooled
dosages into a suitably sized volumetric flask of no less than 25 n L :o generate
an analyte concentration that is within the valic at: d linear range. Di ut : to volume
with Diluent, and sonicate for 10 min.
7.5.4 For chewable gels (gummies), homogenize at le: st 10 dosage unit; a scording tc
the procedure outlined in D-793 Cryogenic Grin ling of Chewable “ic ls. Quickly
weigh a portion of the pooled and homogenize d sages into a beak« r. Jse severa
small portions of Diluent to completely trans{2r she sample into a su tably sizec
volumetric flask to gener. ite an analyte concei tr: tion that is withir the validatec
linear range. Dilute to volun e, and sonicate fcr 1) min.
7.5.5 To manage large volum:s, the standard can b initially prepared it a highe:
concentration and further di uted into the linear r nge using Diluen . | quilibrats
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Standard Operating Procedure SOP No | Rev
Choline Determination by HPLC using UV/VIS D-738 5 | Page 5 of8
Spectroscopy
the solution to room temperature prior to performing further dilution.
Dilutions can be made using volumetric glassware and/or adjustable pipettes.
Dilutions can be prepared in HPLC vials
7.5.6 If particulates remain in the final sample preparation, a portion may be centrifuged
at 10,000 rpm for 5 min prior to HPLC analysis. Alternatively, the sample may
be filtered through a 0.45 um membrane discarding the first 3-4 mL.
8.0 Chromatographic Conditions
8.1 Gradient-Multistep
8.1.1 Time %A %B
0.00 98 2
6.00 98 2
6.1 20 80
8.1 20 80
14.00 98 2
8.1.2 Column- Luna C18(2), 5um, 100 A, 4.6mm x 250mm or equivalent
8.1.3. Flow Rate- 1.0mL/min
8.1.4 UV detection- 194nm
8.1.5 Injection volume- 20uL
8.1.6 | Column Temperature- 30°C
8.1.7 Recommended 3-D Spectral Range- 190nm to 250nm
8.1.8 Retention Time
8.1.8.1 Choline ~ 2.8 min
8.2 Recommended Sequence
8.2.1 Make at least 2 injections of the diluent.
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Spectroscopy
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 six (6) samples and
at the end of the run.
8.3. System Suitability
8.3.1 The %RSD of five (5) injections of Working Standard is NMT 5.0%.
8.3.2 The %RSD of all injections of Working Standard is NMT 5%.
8.4 Column Wash and Storage
8.4.1 Wash the column with H2O/ACN (90/10) at ImL/min for at least 15 min.
8.4.2 Wash the column with H2O/ACN (50/50) at 1 mL/min for at least 15 min.
8.4.3 Store the column with H20/ACN (50/50).
9.0 Example calculations
% assay = = x ae : me x = x 100
Ra Sample peak area
Rs Mean standard peak area
Wtsta Weight of 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)
Vspt 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.
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Spectroscopy
LA Label amount in mg per dose or 1 for raw materials
10.0 Example Chromatography and UV Spectrum
10.1. Blank
Blank
S
sr
RN
°
er
8.2 ;
967.3
628.95
-407 . :
05115225335 445 555 665 7 75 8 85 9 95 1010.5 11 11.5 12 12.5 13 13.5 14
Time [min]
10.2. Standard
22AS056
U oa 2
633.2
257.2
etartratiB
enilohC
967.3 05 115 2253354455 55 6 65 775 8 85 9 95 1010.5 1111.5 12 12.5 13 13.5 14
Time [min]
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Standard Operating Procedure SOP No | Rev
Choline Determination by HPLC using UV/VIS D-738 5 | Page 8 of 8
Spectroscopy
10.3. Finished Product Sample
220254
10004
900+
800+
700;
> 600;
= 500+
400+
300;
200+
1004
933.2
647.2
etartratiB
enilohC
477
aL
05115225335 4455 55 665 775 8 85 9 95 10105 11 11.5 12 12.5 13 13.5 14
Time [min]
10.4 UV Spectrum
Choline Bitartrate - 2.755 (2022-10-28 17-44-05-04-00-02-r006.dx)
100
90-
80-4
70+
60-
& 50+
40+
3100--4 TM\
20-1
Om obo ako abo 2e0~CaO~C«O~«AO~=«OSCSBO.4dO~=«COSCO|SCAG SOS «820 540) 580) 880600
nm
11.0 Revision History
| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 1 | 08/19/13 | New 4 ee tBoi epnrneisaeln tr emveitehw:o du pSdOaPte sdt rSuOctPur Feo. rmat. Up,d ated content to conform 16-0025 Nechang’ | 13-705 | B. Johns |
| 3 | 02/04/19 | Scheduled review: updated responsibilities and corrected typos. Update to reflect current practices and for clarity. Simplify mobile . ee Fnhcalsued inpgre p.B eAMdUdSt rientEe nsttiaonnd atridm.e .N aArdrdo ws ytshtee rma snugitea bifloirty a reiqui rmeamtecnht.s COmeU172 S. Bassani Add column wash and storage. Removed unnecessary information and align with current SOP format, add instruction to follow test details containing product | 19-0113 | J. Maignan |
| 5 | 05/17/23 | specific sample preparation. Added specific sample prep instructions CC- for different dosage forms. Added example chromatography and spectrum. Changed logo. | 23-0225 | S. Sassman |