Identification by Mass Spectroscopy

Original Document

Scanned document (image-only PDF)

Extracted Text

Searchable text extracted from PDF

1.0 Purpose 
 
 The purpose of this procedure is to describe the method and conditions for the qualitative
 
 analysis of defined or purified chemicals by using LC/MS and MS. 
 
 2.0 Scope 
 
 This method is intended to identify defined or purified chemicals by using their specified
 
 molecular weight. Due to each chemical’s specific molecular weight, this method will be
 
 directly used without further validation by reference standards. The method and conditions will
 be listed in detail unless it is otherwise stated. 
 
 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 QC Laboratory Management and/or Analytical Development
 
 to keep this procedure current with latest Ion Labs practices. 
 
 4.0 Definitions 
 
 4.1 MS — Mass Spectroscopy 
 
 4.2 HPLC — High Performance Liquid Chromatography 
 
 4.3. LC/MS - Liquid Chromatography / Mass Spectroscopy 
 
 4.4 ESI-— Electrospray Ionization 
 
 4.5 SIM — Selected Ion Monitoring 
 
 4.6 M+1 — This is the mass most likely to be seen in positive mode. This correlates to the
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev No Page 
 Identification by Mass Spectroscopy D-746 2 of5 
 
 mass of the product plus | as charged molecules fly through the quadrapole.
 
 4.7 M-1-— This is the mass most likely to be seen in negative mode. This correlates to the
 mass of the product minus 1 as charged molecules fly through the quadrapole.
 
 4.8 M/Z — Mass to charge ratio; This corresponds to the mass divided by the overall charge
 
 of the molecule being analyzed. Since the quadrapole will deflect ions with a +2 charge
 twice as heavily, it will be interpreted as M+2/2 by the software. Consequently ions
 
 with a +3 charge will be interpreted as M+3/3. This effect is seen with -2, -3 etc.
 
 charges as well. 
 
 4.9 QC -— Quality Control 
 
 5.0 References 
 
 None 
 
 6.0 Supplies and Equipment 
 
 6.1 Supplies and Glassware 
 
 6.1.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa. 
 
 6.1.2 1L mobile phase container 
 
 6.1.3 Volumetric flask, 500mL, 100mL, 50mL, 25mL 
 
 6.1.4 Beaker, 50mL, 100mL 
 
 6.1.5 Pipette tips, 1OmL, ImL, 200uL 
 
 6.1.6 Eppendorf Tubes, 1.5mL, 1.7mL or 2.0mL 
 
 6.1.7 Weigh boats 
 
 6.1.8 Plastic luer lock syringe, 10mL, 6mL or 3mL 
 
 6.1.9 0.2 or 0.45um 25mm Nylon syringe filters 
 
 6.1.10 0.2 or 0.45um 25mm HDPE syringe filters 
 
 6.2 Equipment 
 
 6.2.1 Agilent 1100 Series HPLC with DAD detector 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev No Page 
 
 Identification by Mass Spectroscopy D-746 3 of 5 
 
 6.2.2 Agilent 6120 MS with ESI source, MSD detector 
 
 6.2.3 Analytical Balance 
 
 6.2.4 Vortex 
 
 6.2.5 Stir Plate 
 
 6.2.6 Eppendorf Centrifuge 
 
 6.2.7 Wrist action shaker 
 
 6.2.8 Pipette 10mL, ImL, 200uL, 10uL 
 
 7.0 Method Parameters 
 
 7.1 HPLC - If separation is required to identify multiple component raw materials the
 
 following parameters need to be defined: (Note: use accurate and up-to-date tune file)
 
 7.1.1 Column type 
 
 7.1.2 Mobile phase 
 
 7.1.3 Dissolution Buffer 
 
 7.1.4 Column temperature 
 
 7.1.5 Wavelength (optional) 
 
 7.1.6 Gradient (optional) 
 
 7.1.7. Injection volume 
 
 7.1.8 Flow rate 
 
 7.1.9 Run time 
 
 7.1.10 Sample concentration 
 
 7.1.11 MS polarity 
 
 7.1.12 Scan/SIM settings 
 
 7.2 Direct MS Injection - If no separation is required to identify raw materials the following
 parameters need to be defined: (Note: use accurate and up to date tune file)
 
 
 

[SOP 

 Standard Operating Procedure SOP No | RevNo | Page 
 Identification by Mass Spectroscopy D-746 3 4 of 5 
 
 7.2.1 Injection volume 
 
 7.2.2 Flow rate 
 
 7.2.3 Sample concentration 
 
 7.2.4 Wavelength (optional) 
 
 7.2.5 MS Polarity 
 
 7.2.6 Scan/SIM settings 
 
 8.0 Sample Preparation 
 
 Samples for identification should be dissolved in Mobile Phase or another suitable
8.1 solvent or solvent mixture. Most analytes will generate a suitable response at a

 concentration of 0.1 mg/mL. To manage large volumes the sample can be initially
 
 dissolved in a smaller volume that is within the solubility range and a portion further
 diluted to bring the analyte concentration into a range to give an adequate signal. The
 
 sample must be filtered or centrifuged before analyzing by MS. 
 
 8.2 All samples must be shaken on a wrist action shaker for 30 minutes at RT in two thirds
 
 their initial volumes then brought up to final volume. 
 
 8.3 For filtration, withdraw 10mL using a 10mL plastic syringe. Attach the appropriate
 hydrophilic or hydrophobic filter to the end of the syringe. Filter and discard the first
 
 0.5mL of sample before collecting. From the collected sample dilute as needed then
 
 add ImL to an HPLC vial for analysis. 
 
 8.4 For centrifugation, add 1.7 to 2.0mL of sample to an Eppendorf tube. Using an
 equivalent tube add an equal volume of water for use as a counter balance. Place the
 
 two tubes in opposing positions in the Eppendorf centrifuge rotor. Centrifuge for 10
 minutes at 3000 RPM. Withdraw an aliquot of supernatant, dilute as needed and add
 
 lmL to an HPLC vial for analysis. 
 
 8.5 If the sample has been previously tested by MS or LC/MS, the system can be set up
 
 using the parameters defined in the electronic MS Parameters for Chemical ID
 spreadsheet located in the F:\Laboratory folder. Otherwise, the following can be used as
 
 a general starting point. For pure compounds, separation by HPLC is not necessary.
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev No Page 
 Identification by Mass Spectroscopy D-746 5 of 5 
 
 8.5.1 Positive Ion Mode 
 
 8.5.1.1 Column: C18 or C8, 150 mm X 4.6 mm, 5 um particle size
 
 8.5.1.2 Mobile Phase A: 0.1% formic acid in water 
 
 8.5.1.3 Mobile Phase B: Acetonitrile 
 
 8.5.1.4 Gradient: Initial 10% B, ramp to 80% B over 15 min 
 
 8.5.1.5 Injection Volume: 10 uwL 
 
 8.5.2 Negative Ion Mode 
 
 8.5.2.1 Column: C18 or C8, 150 mm X 4.6 mm, 5 um particle size
 
 8.5.2.2 Mobile Phase A: 20 mM ammonium acetate in water 
 
 8.5.2.3 Mobile Phase B: Acetonitrile 
 
 8.5.2.4 Gradient: Initial 10% B, ramp to 80% B over 15 min 
 
 8.5.2.5 Injection Volume: 10 uwL 
 
 9.0 Criteria 
 
 9.1 Chemical or chemicals are positively identified when the corresponding molecular
 
 weight of the expected analyte(s) is identified in the MS chromatogram (taking into
 account M+1, M-1 and M/Z if present) and the other principle detected ions can be
 
 explained. 
 
 10.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 04/21/14 | } New l 09/21/16 Biennial review: Updated responsibilities. Removed form. 16-0848 N. Zhang Update responsibilities. Update formatting. Add general guidance | 14-0335 | B. Johns |
| 2 | 11/26/19 | for analytes that have not been previously tested. Added M+1, M-1 and M/Z. | 19-0910 | S. Sassman |
| 3 | 01/30/23 | Update logo. Update formatting. Revise responsibilities section. CC- | 23-0051 | K. Burris |

 
 

[SOP 

 |988d
 
P 
E 
I 
O 
U 
8 
0 
0 
' 
0 
T 
O 
' 
O 
s 
a 
M 
u 
s 
“ S ~ 
S — 
U B I O d 
/ I 
(Z J I T S (:1A M I e 
 
 ZPaA/sasWqc ‘+O5P0EL O:-Z3EOTN 
 
y y syeorwiay wnisouseyy 
e 
s 
u 
o 
s 
i 
n 
w 
T 
/ 
j O V LP vel 
E N I D e e l 
I 
}Od}9p 
y W 
 , 
 ayejoor 
T U a Y y D 0} IIDIDIN a j o j a n d s w U E A y o 
s p e o L p l o y w i n i u o a y e u l l o y
M e y e u o w y y W N i s o u s e W I G o v e ] u m n i s o u s e y y o r e q i e d s y S W A T O I D Y p o s - S I s s e y A d o o s o n o o d s - H O O O T A D o N s 0 v O I N G U - “ ( ¢ H O “ H O ) H N - O O S H O - H O O O T H N w N I M O W W Y H O F W - j o u r y o W - H O O O H SIMHWOF P I V H O P V - S H 9 0 V P I S Y [ e n u a p y u 0 D