D-1003
Determination of Amino Acids by LC-MS
Original Document
Scanned document (image-only PDF)
Extracted Text
Searchable text extracted from PDF
1.0 Purpose
The purpose of this procedure is to define the method for the determination of amino acids and
similar compounds in raw materials and finished products by LC-MS.
2.0 Scope
This procedure applies to the determination of glycine, proline, methionine, hydroxyproline,
glutamine, y-aminobutyric acid, lysine, and ornithine in the QC laboratory at Ion Labs.
3.0 Responsibility
31 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.
ao It is the responsibility of QC Laboratory Management and/or Analytical Development to
keep this procedure aligned with current practices.
4.0 Definitions
4.] LC-MS — Liquid chromatography — mass spectrometry
4.2. QC — Quality control
4.3 ACN - Acetonitrile
4.4 PVDF - Polyvinylidene fluoride
4.5 H20 — Deionized water (>18MQ-cm)
4.6 GLY -Glycine
4.7 PRO — Proline
[SOP D-100 | Page 2 of 13]
Standard Operating Procedure SOP No Rev Page
Determination of Amino Acids by LC-MS D-1003 3 2 of 13
4.8 MET -— Methionine
4.9 HPR- Hydroxyproline
4.10 GABA -—y-Aminobutyric acid
4.11 GLUT -— Glutamine
4.12 LYS-—Lysine
4.13. ORN —- Ornithine
4.14 SER - Serine
5.0 References
| PRTCL-21-0017, Protocol, Validation of an Analytical Method for the Determination of
Glycine, Proline, Hydroxyproline, and Methionine
5.2 PRTCL-21-0055, Protocol, Validation of a Method for the Determination of GABA by
LC-MS
ee, PRTCL-22-0023, Protocol, Validation of a Method for the Determination of Glutamine
by LC-MS
5.4 PRTCL-23-0018, Protocol, Validation of a Method for the Determination of Amino
Acids and Similar Compounds by LC-MS
6.0 Supplies
6.1 Chemicals
6.1.1 Reference standards for the target analytes
6.1.2 — Isotopically labelled amino acid standards
6.1.2.1 L-glutamine-'3Cs,!5No;_— Glycine-!8C2,!°N; — L-lysine-!?C,!°No;
L-methionine-!3Cs,!>N; L-proline-!3Cs,'5N; L-serine-!3C3,!5N
6.1.3 ACN (LC-MS grade)
6.1.4 Ammonium formate (LC-MS grade)
[SOP D-100 | Page 3 of 13]
Standard Operating Procedure SOP No Rev Page
Determination of Amino Acids by LC-MS D-1003 3 3 of 13
6.1.5 Formic acid (LC-MS grade)
6.1.6 Formic acid (HPLC grade)
6.1.7. Methanol (HPLC grade)
6.2 Glassware and Disposables
6.2.1 Volumetric glassware as required for standard and sample preparations
6.2.2 HPLC vials, 2mL with screw-cap enclosures and septa
6.2.3 Tips for adjustable pipettes
6.2.4 0.45 um nylon or PVDF syringe filters
6.2.5 10-mL plastic syringes
6.3 Equipment
6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, and column
oven
6.3.2 Agilent Ultivo mass spectrometer using MassHunter software
6.3.3 Analytical balance
6.3.4 Sonicator
6.3.3 Wrist action shaker
6.3.6 Adjustable pipette and tips
7.0 Preparation of Mobile Phase, Extraction Solvent, Standards, and
Samples
7.1 Mobile Phase A (15 mM ammonium formate + 15 mM formic acid in ACN-H20 82-18)
GA 100% Mobile Phase A is used for GABA, GLUT, GLY, HPR, MET, and PRO.
7.1.2 Transfer 180 mL of H20 to a suitable container.
7.1.3 Add 0.95 g of ammonium formate (LC-MS grade).
[SOP D-100 | Page 4 of 13]
Standard Operating Procedure SOP No Rev Page
Determination of Amino Acids by LC-MS D-1003 3 4 of 13
7.1.4 Add 0.57 mL of formic acid.
7.1.5 Add 820 mL of ACN, and mix well.
7.2. Mobile Phase B (15 mM ammonium formate + 80 mM formic acid in ACN-H20 75-25)
Fidel 100% Mobile Phase B is used for LYS and ORN.
7.2.2 Transfer 250 mL of H20 to a suitable container.
2.3 Add 0.95 g of ammonium formate (LC-MS grade).
7.2.4 Add 3.0 mL of formic acid.
7.2.5 Add 750 mL of ACN, and mix well.
Ie Extraction Solvent
7.3.1. Transfer 100 mL methanol to a suitable container.
7.3.2. Add 10.0 mL of formic acid (HPLC grade).
7.3.3 Add 900 mL of H20, and mix well.
7.4 Internal Standard Stock Solution (0.1 mM)
7.4.1. Prepare 40% methanol solution (scale as necessary).
741.1 Transfer 300 mL of H20 to a suitably-sized beaker.
7.4.1.2 Add 200 mL of methanol, and mix well.
7.4.2 Prepare a solution containing a mixture of each isotopically labelled internal
standard at about 0.1 mM in 40% methanol.
74.2.1 If using Cambridge Isotope Laboratories part number MSK-CAA-1,
prepare the Internal Standard Stock Solution as follows:
7.4.2.1.1 Use several 5 mL portions of 40% methanol solution to
dissolve and completely transfer the contents of the
Canonical Amino Acid Mix standard into a 25-mL
volumetric flask.
[SOP D-100 | Page 5 of 13]
Standard Operating Procedure SOP No | Rev Page
Determination of Amino Acids by LC-MS D-1003 3 5 of 13
7.4.2.1.2 Dilute to volume using 40% methanol solution.
7.4.3 Store the solution at -20°C.
7.4.4 Equilibrate to room temperature, and mix well prior to use.
7.5 Working Internal Standard
75.1 Transfer 0.625mL of the Internal Standard Stock Solution to a 25-mL
volumetric flask.
7.5.2 Dilute to volume using Mobile Phase, and mix well.
7.5.3. Store the solution at -20°C.
7.5.4 Equilibrate to room temperature, and mix well prior to use.
Note: Only prepare standards for analytes to be quantified.
7.6 Stock Standard A (250 mcg/mL PRO, HPR, MET, and GABA)
7.6.1 Accurately weigh and transfer about 25 mg each of PRO, HPR, MET, and
GABA reference standards into a 100-mL volumetric flask.
7.6.2 Dissolve in and dilute to volume with H20.
7.7 Stock Standard B (500 mcg/mL GLUT, LYS, and ORN)
7.7.1. Accurately weigh and transfer about 25 mg each of GLUT, LYS, and ORN
reference standards into a 50-mL volumetric flask.
7.72 Dissolve in and dilute to volume with H20.
7.8 Stock Standard C (500 mcg/mL GLY)
7.8.1 Accurately weigh and transfer about 25 mg of GLY into a 50-mL volumetric
flask.
7.8.2 Dissolve in and dilute to volume with H20.
79 Intermediate Standard (2 mcg/mL PRO, HPR, MET, and GABA; 6 mcg/mL GLUT, LYS,
and ORN; 20 mcg/mL GLY)
[SOP D-100 | Page 6 of 13]
Standard Operating Procedure SOP No | Rev Pa
* ° e ° ° ge
Determination of Amino Acids by LC-MS D-1003 3 6 of 13
7.9.1 Transfer 2.0 mL of Stock Standard A to a 250-mL volumetric flask.
7.9.2 Add 3.0 mL of Stock Standard B.
7.9.3 Add 10.0 mL of Stock Standard C.
7.9.4 Dilute to volume with H20.
7.10 Working Standard (80 ng/mL PRO, HPR, MET, and GABA; 240 ng/mL GLUT, LYS,
and ORN; 800 ng/mL GLY)
7.10.1 Transfer 2.0 mL of Intermediate Standard to a 50-mL volumetric flask.
7.10.2 Dilute to volume with Mobile Phase.
7.10.3 Combine 0.4 mL of the Working Standard with 0.4 mL of the Working Internal
Standard in an HPLC vial, and vortex to mix.
7.11 Sample Preparation (5 mg/mL)
Fel lel 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.
FALL2 The default sample weight is 500 mg. Finished products with large dosage units
and/or very small amount of target analyte may require larger sample size. For
raw materials or finished products with a very large amount of target analyte,
the sample size may be decreased with a minimum recommended weight of 50
mg.
7.11.3 Ensure that the sample is homogeneous prior to weighing.
7.11.3.1 For capsules, combine the fill material from at least 10 dosage units
and homogenize in a mortar and pestle if necessary.
7.11.3.2 For tablets, combine at least 10 dosage units and homogenize in a
mortar and pestle.
[SOP D-100 | Page 7 of 13]
Standard Operating Procedure SOP No Rev
Page
Determination of Amino Acids by LC-MS D-1003 3 7 of 13
7.11.3.3. For chewable gels (gummies), homogenize at least 10 dosage units
as outlined in D-793.
7.11.4 Transfer 500 mg of sample to a 100-mL volumetric flask.
dak Add about 65 mL of Extraction Solvent.
7.11.6 Sonicate 5 min.
rin ry, Shake on a wrist-action shaker for 20 min.
7.11.8 Dilute to volume with Extraction Solvent.
7.11.9 If the sample is laden with particulate matter, filtration through a 0.45 um
membrane (discarding 2 — 3 mL before collecting the sample for further
dilution) may be necessary prior to further dilution of the sample.
TAZ Working Sample Preparation
Vale The linear range of the method for each analyte is listed below. The working
sample preparation must be within the linear range of the method.
7.12.1.1 PRO, HPR, MET, and GABA: 25 — 125 ng/mL.
7.12.1.2 GLUT, LYS, and ORN: 80 — 400 ng/mL.
7.12.1.3. GLY: 250-1250 ng/mL.
M dues Dilute the Stock Sample to target concentration for each amino acid. Perform
the final dilution using Mobile Phase. If multiple dilutions are required, perform
intermediate dilutions using H20.
FAl2d Filter through a 0.45 um membrane (discarding 2 — 3 mL before collecting the
sample for analysis). If the stock sample was filtered, filtration of the working
sample is not required.
7.12.4 Combine 0.4 mL of the Working Sample with 0.4 mL of the Working Internal
Standard in an HPLC vial, and vortex to mix.
7.13 Instrument Method Parameters
[SOP D-100 | Page 8 of 13]
Standard Operating Procedure SOP No Rev Page
Determination of Amino Acids by LC-MS D-1003 3 8 of 13
7.13.1 Place an HPLC vial containing a blank (diluent) in position P1-A1.
7.13.2 Place an HPLC vial containing the working standard in position P1-A2
7.13.3 Analyte that require different Source Parameters (Section 7.13.8.1.4) must be
analyzed with separate acquisition methods
7.13.4 Column: Kinetex HILIC, 2.6 um, 2.1 mm x 100 mm or equivalent
7.13.5 Sampler
7.13.5.1 Injection Volume: 4 pL
Tel 30d Enable Needle Wash: Selected
Jil 3uds3 Mode: Flush Port
734 Time: 3 sec
Tel Bidd Sample Flush-Out Factor: 5.0 times injection volume
7.13.5.6 Overlapped Injection Mode: Off
7.13.6 Binary Pump
7.13.6.1 Flow Rate: 0.4 mL/min
7.13.6.2 Mobile Phase: Isocratic
7.13.6.3 Stoptime: 5.5 min
7.13.7. Column Oven
FASid Temperature: 35 °C
7.13.8 QQQ
71381 Acquisition
7.13.8.1.1 Scan type: MRM
7.13.8.1.2 Polarity: Positive
7.13.8.1.3 Acquisition Parameters
[SOP D-100 | Page 9 of 13]
Standard Operating Procedure SOP No Rev Page
Determination of Amino Acids by LC-MS D-1003 3 9 of 13
Analyte Int Std | MSI Res ae a a : Dwell (ms) an CE (V)
GLY GLY-IS Unit 76.0 Unit 48.0 200 4] 0
GLY-IS N/A Unit 79.0 Unit 50.0 200 4] 0
PRO PRO-IS Unit 116.0 Unit 70.0 75 70 10
PRO-IS N/A Unit 122.0 Unit 75.0 75 70 10
HPR SER-IS Unit 132.0 Unit 68.0 180 80 14
SER-IS N/A Unit 110.0 Unit 63.0 75 60 7
MET MET-IS Unit 150.0 Unit 56.0 110 88 12
MET-IS N/A Unit 156.0 Unit 60.0 110 88 12
GABA _ | GLUT-IS Unit 104.0 Unit 87.0 60 42 6
GLUT | GLUT-IS Unit 147.1 Unit 84.0 160 60 15
GLUT-IS N/A Unit 154.4 Unit 89.0 160 60 15
LYS LYS-IS Unit 147.0 Unit 84.0 360 65 15
LYS-IS N/A Unit 155.0 Unit 90.0 360 65 15
ORN LYS-IS Unit 133.0 Unit 70.0 460 60 18
7.13.8.1.4 Source Parameters
Gas Gas Nebulizer | Sheath | Sheath | Capillary | Nozzle
Analytes Source | Temp Flow Pressure | Temp Flow Voltage | Voltage
(°C) | (L/min) (psi) (°C) (L/min) (V) (V)
GLY, GLY-IS,
GLUT, GLUT- | AJS ESI | 300 8 50 350 12 2500 100
IS, GABA
PRO, PRO-IS,
MET, MET-IS, | AJS ESI | 300 8 25 350 12 4500 500
HPR, SER-IS
ORN, LYS,
LYS-IS AJS ESI | 300 8 25 350 12 3500 350
7.14 Retention Times
Analyte Retention Time (min)
MET, MET-IS 1.8
HPR 2.9
SER-IS 2.9
PRO, PRO-IS 3.0
GLY, GLY-IS 3.2
ORN 33
GLUT, GLUT-IS 3.5
LYS, LYS-IS 3.6
GABA 4.0
[SOP D-100 | Page 10 of 13]
Standard Operating Procedure SOP No Rev Page
Determination of Amino Acids by LC-MS D-1003 3 10 of 13
7.15 Recommended Sequence
7.15.1 Make at least 3 injections of Diluent.
7.15.2. Make 5 injections of the Working Standard.
7.15.3. Make a single injection of each Working Sample.
7.15.4 Make a single injection of the Working Standard after every six samples and at
the end of the run.
7.16 System Suitability Requirements
7.16.1 The %RSD for five consecutive injections of Working Standard is NMT 3%.
7.16.2 The %RSD for all injections of Working Standard is NMT 5%.
7.16.3 No significant (>0.5%) interference is present in the diluent injection.
7.17. Column Storage
7.17.1 Store the column in Mobile Phase.
7.18 Column Deep Clean and Storage
7.18.1 Cleaning Solution (100 mM ammonium formate in ACN-H20 10-90)
7.18.1.1 Transfer 450 mL of H20 to a suitable container.
7.18.1.2 Add 3.854 g of ammonium formate (ACS reagent grade).
7.18.1.3 Add50 mL of ACN, and mix well.
7.18.2 Deep clean the column at 0.2 mL/min with Cleaning Solution for at least 60
min.
7.18.3. Wash the column at 0.2 mL/min with H2O/ACN (90/10) for at least 20 min.
7.18.4 Wash the column at 0.2 mL/min with Mobile Phase for at least 20 min.
7.18.5 Store the column in Mobile Phase.
7.19 System Wash
7.19.1 Perform system wash if the instrument will not be used for more than one week,
7.19.2 Remove the column.
[SOP D-100 | Page 11 of 13]
Standard Operating Procedure SOP No | Rev
Page
Determination of Amino Acids by LC-MS D-1003 3 11 of 13
7.19.3 Purge all lines with H2O/ACN (50/50).
7.19.4 Open the instrument method.
7.19.5 Download the method to the instrument.
7.19.6 Pump H20/ACN (50/50) through the system, including the MS detector, for at
least 30 min.
7.19.7 Put the instrument into standby mode.
8.0 Example Calculation
Ru WtsaxP SS Vepl
% assay = RL x x 100.
Vsta Splwt LA
Sample relative response
Mean standard relative response
Weight of reference standard in mg
Volume of the standard preparation accounting for dilutions in mL
Purity of the reference standard in decimal format
SS Serving size: Weight of a single dosage unit in mg or | for raw materials.
Splwt Sample weight in mg
Vspl Volume of the sample preparation accounting for dilutions in mL
LA Label amount in mg per dose or 1 for raw materials
[SOP D-100 | Page 12 of 13]
Standard Operating Procedure SOP No | Rev Page
Determination of Amino Acids by LC-MS D-1003 3 12 of 13
9.0 Example Chromatograms for Target Analytes
+ MRM (76.0 -> 48.0) glycine GLY-*
2 x1034 2.22 min.
= 44 25572,
pat 34
24
4
T | T T q T T T T i T T al T T T T T Ll T T q T 7
02 O04 06 08 i 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time (min)
+ MRM (127.1 -> 84.0) glutamine GLUT-1
4g% xxi19 0 $4i 23.052.min.
oO 064
o44
0.24 0
T T T T ii y T T T T T T T T T T T T T t T T T T
02 04 06 08 i 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time (min)
+ MRM (104.0 -> 87.0) gaba GABA-1
& 108 $.03 min
Een 703420,
tab
0.54 0
T T T T qT T T T i) T T T T T T T T T if i T y T T
02 04 06 O08 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time (min)
+ MRM (150.0 -> 56,0) methionine MET-PRO-HPR-7
@
x103
3
4
1
2
.7
i
6f,emian.
cS 3
04
T T T T T T T T T T T T T T T T i T i T T T 1 qT
02 04 06 O8 4 72 14 16 18 #2 22 24 26 28 3 32 34 36 28 4 42 44 46 48
Acquisition Time (min)
+ MRM (116.0 -> 70.0) proline MET-PRO-HPR-1
£ x104] Tt min
tA
o T T UJ T T T T y T T
o2 of o6 of + v2 14 16 «18 2T 2T2 24 2T6 DTe 3 32 34 326 28 4 42 446 46 48
Acquisition Time {min}
+ MRM (132.0 -> 68.0) hydroxyproline MET-PRO-HPR-1
2 x192 7 2.89836 2m.i5n.
=,
ial \
° T T T T T q T T T T T T T 7 il T T T T T T i J T
02 04 O06 O8 1% 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time {min}
+ MRM (147.0-> $4.0) lysine SP-LYS-ORN
2 x08] | | 2.76 min
3 gos| | 7191.2
0.025544 ee twtinn. ganyceal
a- T q T T T T T T T T T T T T T T T T T T T qT 7 T
02 04 06 O08 4 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time imin}
+ MRM (133.0 -» 70.0) ornithine LYS-ORN-?
2 x02 2.26.min.
& 4
3 NN
2
1
oo of o6 o@ . v2 it we ve 2 22 23 2e 2e 3 32 2 ge se 4 go se ae 48
Acquisition Time (min)
[SOP D-100 | Page 13 of 13]
Standard Operating Procedure SOP No Rev Page
Determination of Amino Acids by LC-MS D-1003 3 13 of 13
10.0 Example Chromatograms for Internal Standards
+ MRM (79.0 -> 50.0) alycine-IS SP-GLY
2 x103 3.19 min.
Fe - 6547.7
w ,sae agua ama 4
02 04 06 08 } 1i2 1q4 i 16 1T8 2i 22 24 26 28 3 32 34 36 38 4 42 44 46 ae
Acquisition Time (min)
+ MRM (154.4 -> 89.0) glutamine-IS SP-GLY
2 xt04 3.48 min.
cary
172810.
6
] { i i | i J T i ij ] ] 1 [J | i ! J ! q i ij Ty
02 04 #06 08 12 14 #16 18 #2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time (min)
+ MRM (156.0 -> 60.0) methionine-IS MET-PRO-HPR-1
2 x05 1.76 min.
2 08 304280.
a be. T i T q a qT T T y T q T T y T i i q T i J T q
02 04 06 08 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time (min)
+ MRM (122.0 -> 75.0) proline-IS MET-PRO-HPR-1
& 105] 3.02 min.
2 2.
1225500,
O : AA.
[ae ete ie aie le le * aly Ra De lor la Se les a, de ale ae dy ee
02 04 06 O08 4 #12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48
Acquisition Time (min)
+ MRM (110.0 -> 63.0) serine-IS MET-PRO-HPR-1
2 x1034 2.87 min,
3 4 41925,
C 254 aN
0 ] ] ] ! t i T ] T ] ij T i ] ] T T T i] q q T i
02 04 06 08 2 14 168 #18 2 22 24 286 28 2 32 34 36 38 4 42 44 46 48
Acquisition Time (min)
+ MRM (155.0 -> 90.0) lysine-IS LYS-ORN-1
2MA x10°8 ] 3m ii
04 —
_ ho ok ls oe In es le le a ie ie ae ae ale ie ce eee al
02 04 06 08 12 14 16 18 2 22 24 26 28 3 32 24 36 38 4 42 44 46 48
Acquisition Time (min)
10.0 Revision History
| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 09/28/21 | New procedure. N/A S. Sassman Add GABA as a target analyte. Adjust source parameters for I 12/06/21 proline, methionine, and hydroxyproline to improve linearity. CC-21-0480 S. Sassman Minor edits for clarity. | - | - |
| 2 | 01/19/23 | Add glutamine as a target analyte. CC- Change calibration approach from standard additions to internal | 23-0034 | S. Sassman |
| 3 | 10/26/23 | standard, add lysine as an analyte, and combine with D-1005 CC- (add ornithine as an analyte), change extraction solvent. | 23-0527 | S. Sassman |