Guideline for Lc-Ms Users

GUIDELINE FOR LC-MS USERS

Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). LC-MS is a powerful technique that has very high sensitivity and selectivity and so is useful in many applications.

SAIF CSIR-CDRI, Lucknow is providing only qualitative analysis service to our SAIF Users.

Stepwise experiments

1. HPLC/LC-MS Method development: The method is the collection of conditions in which the HPLC and MS operates for a given analysis Or Method development is the process of determining what conditions are adequate and/or ideal for the analysis required.

2. LC-ESI-MS analysis: Liquid chromatography-mass spectrometry (LC-MS) as a widely used technique for identification and quantification of molecules/compounds/analytes separated by liquid chromatography.

It provides separation of compounds and detection by MS (provide molecular weight of compounds).

3. LC-ESI-MS/MS analysis: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is technique in which fragmentation of molecules/compounds/analytes use to identify/ confirm the chemical structure.

Note-The charges will be for acquiring and providing you the data, not for data analysis.

User can submit samples under 1-3 options one by one and charges will be calculated by software.

1. Register on www.saiflucknow.org

2. Login in your account.

3. Go for Submit sample.

4. Select/ SAIF-Liquid Chromatography–Mass Spectrometry (LC-ESI or APCI-MS)

5. Fill the necessary information and calculate the charges for selected analysis. 2

LC-MS/MS Instrument

Developed for integrated UPLC MS/MS qualitative and quantitative applications, the Waters ACQUITY® TQD features the highest levels of tandem quadrupole MS selectivity, robustness, speed, and accuracy.

Specification:

1. ZSprayTM dual-orthogonal API source

2. Mass scan range: m/z 50-2000 Th

3. Acquisition speed: 10 scan/second

4. Polarity switching(ES+/ ES-): 20 ms 3

5. ESCi mode switching: 20 ms

6. MRM Sensitivity : 1 pico gram (Reserpine S/N 2000:1)

Application notes: http://www.waters.com/waters/en_IN/ACQUITY-TQD/nav.htm?cid=514467&locale=en_IN

http://www.waters.com/webassets/cms/library/docs/720002329en.pdf 4

INFORMATION REQUIREMENTS FOR ANALYSIS

S.N0. Analysis Information require for analysis 1. HPLC/LC-MS Nature of sample: plant extracts/chemical reaction Method products/pure isolated compound from column Or development Fraction of plant extract: like hexane, chloroform, ethyl acetate etc. Solubility: Acetonitrile, Methanol or water Expected : Molecular weight of analytes 2. [LC-ESI-MS analysis] 1. Column: ODS-2, 250 X 4.6, 5 µm

or 2. Mobile Phase: A: [CH3CN]

Liquid B: [5 mM AcNH4] chromatography- 3. HPLC Program: 0-30 min; 80-30%B, 40min; 5%B mass spectrometry Isocratic upto 60min (LC-MS) 4. Flow Rate: 1.0 mL/min 5. Injection Volume: 25μL 6. Concentration of sample: 5 mg/mL 7. MS Ionization Mode: ESI (±) or APCI (±) 8. UV absorption: 254 nm

3. [LC-ESI-MS/MS] 1. Column: ODS-2, 250 X 4.6, 5 µm

analysis 2. Mobile Phase: A: [CH3CN]

or B: [5 mM AcNH4] Liquid 3. HPLC Program: 0-30 min; 80-30%B, 40min; 5%B chromatography- Isocratic upto 60min mass spectrometry (LC-MS/MS) 4. Flow Rate: 1.0 mL/min 5. Injection Volume: 25μL 6. Concentration of sample: 5 mg/mL 7. MS Ionization Mode: ESI (±) or APCI (±) 8. UV absorption: 254 nm 9. Peak list: Retention time: 3.51 min (m/z 395) 5.43 min (m/z 275) 6.86 min (m/z 520) Note- without require information analysis can’t be performed 5

Example: sample preparation

Keywords: Extraction; Liquid-liquid extraction;

1. Weight out 10 mg of solvent free plant extract’s fraction in eppendorf tube.

2. Label with sample code and properly packed in a thermocol box.

3. Send for analysis along with all necessary details. 6

Online information through www.saiflucknow.org

Register in Website Registration Form

Login by Username and Password

Click on Submit sample 7

Select facility and No. Of samples

Fill the application form

HPLC Method details 8

Submit sample

Fill draft details and Submit lot

After submit lot, please view lot status or check your email to view lot details. 9

Application form for offline sample submission

To Head Sophisticated Analytical Instrument Facility CSIR-Central Drug Research Institute P.O.No. 173, Lucknow-226031, UP (India)

Reference : Sample Analysis

Dear Sir,

Please accept the following sample for the analysis, the details are underneath

Draft Details Draft No. Bank Branch Draft Date Amount

S# Sample Qty. in mg Solubility Analysis Mass Range 1

Note :

1. Please make draft in favour of the Director, Central Drug Research Institute, Lucknow-226031. 2. Separate samples should be sent for different analysis. 3. All concern persons, research fellows and students are advised to send their application and samples through their supervisors or Head of Department. The request should be made only on University/College/Institute letter head.

Signature

Name of supervisor...... Email-ID: ...... Tel/Mobile No......

Additional Information:

1. HPLC/LC-MS Method development:

Nature of sample: ………………………………..

Solubility: …………………………………………….

Expected (between; m/z 150-2000): ………………………………….

2. [LC-ESI-MS analysis] Liquid chromatography-mass spectrometry (LC- MS)

SAIF/Lot Number of previously LC-MS developed method:………………………………. (Acquisition Experiment Report) Or [1] Name of column: [2] Mobile Phase: [3] HPLC Program: [4] Flow Rate: [5] Injection Volume: [6] Concentration of sample: [7] MS Ionization Mode: [8] UV absorption:

3. [LC-ESI-MS/MS analysis] Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

SAIF/Lot Number of previously LC-MS developed method:………………………………. Acquisition Experiment Report and 05 m/z peak list for MS/MS with their retention time. Or [1] Name of column: [2] Mobile Phase: [3] HPLC Program: [4] Flow Rate: [5] Injection Volume: [6] Concentration of sample: [7] MS Ionization Mode: [8] UV absorption: [9] 05 m/z peak list for MS/MS with their retention time 11