D-1006
Determination of Methylcobalamin and Cyanocobalamin by LC-MS
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1.0 Purpose
The purpose of this procedure is to define the method for the determination of methylcobalamin
and cyanocobalamin by LC-MS.
2.0 Scope
This procedure applies to the determination of methylcobalamin and cyanocobalamin in raw
materials and finished products by LC-MS in the QC laboratory at Ion 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 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 LC-MS — Liquid Chromatography — Mass Spectrometry
4.2 QC -— Quality control
4.3. ACN - Acetonitrile
4.4 H20 — Deionized water
5.0 References
5.1 PRTCL-21-0058, Protocol, Validation of an Analytical Method for the Determination of
Methylcobalamin, Cyanocobalamin, and Hydroxycobalamin
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5.2. D-793, SOP, Cryogenic Grinding of Chewable Gels
6.0 Supplies
6.1 Chemicals
6.1.1 Methylcobalamin and/or cyanocobalamin reference standards
6.1.2 ACN (LC-MS grade)
6.1.3. Methanol (HPLC grade)
6.1.4 Ammonium acetate (LC-MS grade)
6.1.5 Ammonium acetate (ACS reagent grade)
6.1.6 Acetic acid (LC-MS grade)
6.1.7. Acetic acid (ACS reagent grade)
6.1.8 H20 (= 18 MQ-cm)
6.2 Glassware
6.2.1 Volumetric glassware as required for standard and sample preparations
6.2.2 HPLC vials, 2mL with screw-cap enclosures and septa
6.3 Equipment
6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, and column
oven coupled with Agilent Ultivo mass spectrometer using MassHunter software
for instrument control and data processing.
6.3.2 Analytical balance
6.3.3 Wrist action shaker
6.3.4 Sonicator
6.3.5 Adjustable pipette and tips
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Standard Operating Procedure
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Cyanocobalamin by LC-MS
7.0 Procedure
7.1 Mobile Phase A (15 mM ammonium acetate + 5 mM acetic acid + 10% ACN)
Fakes Transfer 0.58 g of ammonium acetate (LC-MS grade) to a 500-mL bottle.
Filet Add 50 mL of ACN.
7.1.3 Add 0.143 mL of acetic acid.
7.1.4 Add 450 mL of H2O, and mix well.
FZ Mobile Phase B (15 mM ammonium acetate + 5 mM acetic acid + 46% ACN)
Peay Transfer 0.58 g of ammonium acetate (LC-MS grade) to a 500-mL bottle.
7.2.2 Add 270 mL of H20.
Ths Add 0.143 mL of acetic acid.
7.2.4 Add 230 mL of ACN, and mix well.
7.3 Diluent (30 mM ammonium acetate + 10 mM acetic acid + 10% methanol)
7.3.1 Equilibrate to room temperature before using Diluent.
f pe: Transfer 2.31 g of ammonium acetate (ACS reagent grade) to a 1000-mL bottle.
[3,8 Add 100 mL of methanol.
7.3.4 Add 0.57 mL of acetic acid.
7.3.5 Add 900 mL of H20, and mix well.
7.4 Extraction Solvent (16.5 mM ammonium acetate + 5.5 mM acetic acid + 45% methanol)
7.4.1 Equilibrate to room temperature before using Extraction Solvent.
7.4.2 Transfer 550 mL of Diluent to a 1000-mL bottle.
7.4.3 Add 450 mL of methanol, and mix well.
7.5 Stock Standard (250 mcg/mL)
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7.5.1 The target analytes are susceptible to degradation when exposed to light
and/or heat. Standards and samples must be freshly prepared on same day
as testing to ensure accuracy and reliability of results. Previously prepared
composites are not acceptable. Standards and samples should be prepared in
low-actinic glassware and refrigerated immediately after preparation.
7.5.2 Include only those analytes being quantified in the Stock Standard.
7.5.3 Accurately weigh and transfer about 25 mg of each reference standard into a 100-
mL low-actinic volumetric flask.
7.5.4 Dissolve in and dilute to volume with H20.
7.6 Intermediate Standard (2 mcg/mL)
7.6.1 Transfer 2.0 mL of Stock Standard to a 250-mL low-actinic volumetric flask.
7.6.2 Dilute to volume with H20.
ocr Working Standard (24 ng/mL)
7.7.1 Transfer 3.0 mL of Intermediate Standard to a 250-mL low-actinic volumetric
flask.
7.7.2. Dilute to volume with Diluent.
7.7.3. The Working Standard may be stored at 4°C in the dark for the specified time
depending on the target analyte:
7.7.3.1 Methylcobalamin: 2 days
7.7.3.2. Cyanocobalamin: 3 days
7.8 Stock Sample Preparation (40 mg/mL)
7.8.1 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 of this
method.
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Standard Operating Procedure
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7.8.2 | Homogenize the sample prior to weighing:
7.8.2.1. For tablets, combine at least 10 dosage units and grind in a mortar and
pestle.
7.8.2.2 For capsules, remove the fill material from at least 10 dosage units
and grind in a mortar and pestle.
7.8.2.3. For powders and liquids, turn end-over-end in a container with
sufficient head space to allow thorough mixing.
7.8.3. For chewable gels (gummies), homogenize as outlined in D-793.
7.8.4 The default sample weight is 4 g. Finished products with large dosage units
and/or very small amount of target analyte may require larger sample size. For
raw materials, the sample size may be decreased with a minimum recommended
weight of 50 mg.
7.8.5 Accurately weigh and transfer about 4 g of sample to a 100-mL low-actinic
volumetric flask.
7.8.6 Add about 65 mL of Extraction Solvent.
7.8.7 Sonicate for 2 minutes, accurately timed, with occasional shaking.
7.8.8 Shake for 10 minutes, accurately timed.
7.8.9 Dilute to volume with Extraction Solvent.
7.9 Working Sample Preparation
7.9.1 The target concentration is the concentration of the Working Standard. Use the
label claim and fill weight (for finished products) or theoretical percent assay (for
raw materials) to calculate the dilution of the Stock Sample required to reach the
target concentration. The final sample concentration must be within the range 15
ng/mL — 35 ng/mL.
7.9.2 Prepare the Working Sample in low actinic glassware.
7.9.3 Dilute the Stock Sample to the required target concentration using Diluent.
Multiple dilutions may be required.
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Standard Operating Procedure
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7.9.4 Do not centrifuge. If particulates remain in the final sample, filter through a 0.45
um PVDF or nylon syringe filter discarding a portion before collecting the sample
for analysis.
7.10 Instrument Method Parameters
7.10.1 A vial with Diluent must be placed in position P1-A1.
7.10.2 A vial with Working Standard must be placed in position P1-A2.
7.10.3 Three separate Instrument Methods must be created with identical parameters
except for the Sampler Pretreatment section as outlined below.
7.10.4 Column: Agilent Eclipse Plus C18 RRHD, 1.8 um, 2.1 mm x 50 mm
7.10.5 Sampler
7.10.5.1 Injection Volume: N/A (method uses injector program instead).
7.10.5.2 Sample Thermostat: 12°C (control in Instrument Status pane)
7.10.5.3 Enable Needle Wash: Selected
7.10.5.4 Mode: Flush Port
7.10.5.5 Time: 6 sec
7.10.6 Sampler Pretreatment
7.10.6.1 No Spike
7.10.6.1.1 Draw 8.00 uwL from location “P1-A-1” with 100 uL/min
using default offset.
7.10.6.1.2 Draw 4.00 uL from sample with 100 wL/min using default
offset.
7.10.6.1.3 Wash needle in flushport for 3 sec.
7.10.6.1.4 Mix 12 wL from air with 200 wL/min for 5 times.
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7.10.6.2 Low Spike
7.10.6.2.1 Draw 4.00 uL from location “P1-A-1” with 100 wL/min
using default offset.
7.10.6.2.2 Draw 4.00 uL from sample with 100 uL/min using default
offset.
7.10.6.2.3 Wash needle in flushport for 3 sec.
7.10.6.2.4 Draw 4.00 wL from location “P1-A-2” with 100 uwL/min
using default offset.
7.10.6.2.5 Wash needle in flushport for 3 sec.
7.10.6.2.6 Mix 12 uL from air with 200 uL/min for 5 times.
7.10.6.3 High Spike
7.10.6.3.1 Draw 4.00 uL from sample with 100 uwL/min using default
offset.
7.10.6.3.2 Wash needle in flushport for 3 sec.
7.10.6.3.3, Draw 8.00 wL from location “P1-A-2” with 100 pL/min
using default offset.
7.10.6.3.4 Wash needle in flushport for 3 sec.
7.10.6.3.5 Mix 12 uL from air with 200 uL/min for 5 times.
7.10.7 Binary Pump
7.10.7.1 Flow Rate: 0.35 mL/min
7.10.7.2 Gradient
Time (min) “oA “7B
0.00 100 0
3.00 25 75
3.01 100 0
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Standard Operating Procedure
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Cyanocobalamin by LC-MS
| 5.00 | Cs
7.10.7.3 Stoptime: 5 min
7.10.8 Column Oven
7.10.8.1 Temperature: 35 °C
7.10.9 QQQ
7.10.9.1 Acquisition
7.10.9.1.1 Scan Type: MRM
7.10.9.1.2 MS1 Resolution: Unit
7.10.9.1.3 MS2 Resolution: Unit
7.10.9.1.4 Polarity: Positive
7.10.9.1.5 Dwell Time: 400 ms
7.10.9.1.6 Acquisition Parameters
Analyte ISTD Type Pre(cmu/zr)s or | Pr(ondiuec t | Frag“mVe)n tor | C(VE)
evahoosbalamin = quantifier 678.6 147.1 160 45
qualifier 678.6 358.8 160 21
ted lesbalamin i quantifier 673.2 147.1 140 4S
qualifier 673.2 358.9 140 25
7.10.9.2 Source Parameters
7.10.9.2.1 Ion Source: AJS ESI
7.10.9.2.2 Gas Temperature: 290 °C
7.10.9.2.3 Gas Flow: 5.0 L/min
7.10.9.2.4 Nebulizer Pressure: 25 psi
7.10.9.2.5 Sheath Gas Temperature: 300 °C
7.10.9.2.6 Sheath Gas Flow: 8.0 L/min
7.10.9.2.7 Capillary Voltage (Positive Setpoint): 4000 V
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Standard Operating Procedure SOP No Rev Page
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7.10.9.2.8 Nozzle Voltage (Positive Setpoint): 0 V
7.11 Recommended Sequence
7.11.1 Allow the instrument to equilibrate under initial conditions for at least 15 minutes.
The sample thermostat must be within 2°C of the setpoint before starting the run.
7.11.2 Make at least 3 injections of Diluent using the method for No Spike.
7.11.3 Make 5 injections of the Working Standard using the method for No Spike.
7.11.4 Make a single injection of the Working Sample using the method No Spike.
7.11.5 Make a single injection of the Working Sample using the method for Low Spike.
7.11.6 Make a single injection of the Working Sample using the method for High Spike.
7.11.7 Repeat steps 7.11.4 — 7.11.6 for each sample to be analyzed.
AY: System Suitability Requirements
7.12.1 The %RSD for five consecutive injections of the Working Standard is NMT 5.0%.
The RSD is evaluated for each sample set.
7.12.2 The coefficient of determination (R’) for each standard addition series is NLT
0.990. The coefficient of determination is evaluated for each sample tested.
7.12.3 The quantifier/qualifier ratio for each unspiked sample injection is within + 20%
of the average quantifier/qualifier ratio for the five working standard injections.
7.12.4 No significant (>0.5%) interference is present in the diluent injection.
7.13 Column Wash and Storage
7.13.1 Store the column in Mobile Phase.
8.0 Example calculation
8.1 The calculation may be performed automatically by chromatographic software.
8.2 Plot the data obtained from the unspiked and spiked samples with spike concentration on
the x-axis and peak area on the y-axis.
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Standard Operating Procedure
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Cyanocobalamin by LC-MS
8.3 Perform linear regression of the data to obtain the equation y = mx + b.
8.4 Calculate the amount of target analyte in the sample:
—b x DF X FW
Amino Acid (mg) = 7g
10°" /mg Xm x SW
b y-intercept of the linear regression
DF Dilution factor in mL
FW Unit dose weight (in g for finished products) or 100 (raw materials)
SW Sample weight in g
m slope of the linear regression (mL/ng)
9.0 Example Chromatography
9.1 Methylcobalamin Blank
+ MRM (673.2 > 147.1) methyicobalamin DILUENT
2 x10?)
1.2-
os
08-
06-
0.4-
0.24 0 i i ' i 1 ' i t t j
24 25 26 27 28 29 3 31 3.2 3.3
Acquisition Time (min)
9.2 Methylcobalamin Standard
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Standard Operating Procedure
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Determination of Methylcobalamin and
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Cyanocobalamin by LC-MS
+ MRM (673.2 -> 147.1) methyicobalamin WSTD
x10 ¢ stnuoC
all 2 83 min.
12+ 553.00
{-
08-
06-
0.4-
0.2-
0
235 24 245 25 255 26 265 27 275 28 285 29 295 305 31 315 32 325 33
3
Acquisition Time (min)
9.3. Methylcobalamin Sample
+ MRM (673.2 -> 147.1} methyicobalamin 220007-01-MeC-1
2x10?
3 " 2.84 min
5 14- 672.00
1.2-
j-
0.8-
0.6-
04-
02-
0
24 25 26 27 28 wW 3.2 3.3
3.2 Acquisition Time (min)
9.4 Cyanocobalamin Blank
+ MRM (678.6 -> 147.1) cyanocobalamin DILUENT
8 14
0.9-
0.8-
0.7-
0.6-
05-
04-
0.2-
0.1-
0 T T T T T T T
1.3 1.4 15 1.6 1.7 1.8 1.9 4-h 21 22 23 24
Acquisition Time (min)
9.5 Cyanocobalamin Standard
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Standard Operating Procedure
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Determination of Methylcobalamin and
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Cyanocobalamin by LC-MS
x10 2-
1-
08-
stnuoC
+ MRM (678.6 -> 147.1) cyanocobalamin W/STD
1. ) 3 1. T 4 15 16 22 23 24
Acquisition Time (min)
9.6 Cyanocobalamin Sample
+ MRM (678.6 -> 147.1) cyanocobalamin 210137-01-CyC-1
1.89 min.
$15.00
a
eens
' S 1.6 1.7 1.8 1.9 2.1 eoN 2'2 23 Y 24
Acquisition Time (min)
10.0 Revision History
| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 05/16/22 | New N/A S. Sassman Add direction to follow product specific test details listed in the product profile, add option to modify the default sample weight, add at. ie instruction for homogenization of different dosage forms, add Sasi). Sesetian example chromatography. INV-25-0001 and CAPA-25-0002 were iniated to address the initial low LC-MS result for B12 being due to the use of an aged composite Ashton . re this SOP is updated with new suitability of sample composite aia Lukes requirements to prevent recurrence. | - | - |