GCMS maintenance for drug of abuse screening

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

11 posts Page 1 of 1
Good afternoon,
We have a project of screening drugs of abuse with GC-MS (GC: Agilent 7890, MS: Agilent 5977, Column: HP-MS).
We use the Agilent M2721 protocol (see below) with few modifications:

1) Hydrolysis of 1 mL urine with HCL 12N, 15 min, add the phosphate buffer and use NAOH to bring it to pH 6 after the hydrolysis

2) load 1 mL of the hydrolyzed urine and 1 mL of non-hydrolyzed urine into the Bond elut certify column using a vaccum manifold.

3) Elute a third fraction (containing Morphine and Benzoylecgonin) with 20% 2-propanol + 2% NH4OH + 78% CH2CL2 and derivatize with BSTFA (1% TMCS).

Reconstitution after evaporation is made into 20 uL of EtOAc for the acidic and basic fraction and BSTFA( 1% TMCS) for the third.

This allows us to detect all the desired drugs in SCAN MODE but there is an issue with the liner (Agilent 5190-2293). In order to keep our sensitivity, we have to change it really often, almost every run (20-30 sample's injections).

Is it something normal? Or do we do something wrong?
Would it be another solution in order to not change this liner too often?

Thank you for your help.

----------------------------------------------------------------------------------------

EXTRACTION OF DRUGS OF ABUSE
USING BOND ELUT CERTIFY
ANALYTE NAME/MATRIX
M2721

General Drug Screen
from Urine or Plasma

ANALYTICAL
TECHNIQUE

GC or
GC/FID

PRODUCT/PART NUMBER USED

130 mg Certify
1210-2051 or 1211-3050

section 1 - PRINCIPLE AND MECHANISMS

Basic, acidic and neutral drugs can be retained and selectively eluted using the ion
exchange, polar, and non-polar interactions of the Certify mixed mode sorbant.

section 2 - EXTRACTION METHOD
SAMPLE PREPARATION:

To 2 mL of either urine or plasma add 6 mL of 0.1 M phosphate buffer (pH 6.0).
Mix/vortex.

COLUMN PREPARATION/EXTRACTION:
1. BOND ELUT CERTIFY PREPARATION

2 mL CH3OH; draw through under vacuum.
2 mL 0.1 M phosphate buffer (pH 6.0); draw through under vacuum.
NOTE: Use a low vacuum (2 inches Hg) to prevent drying of sorbent.

2. SPECIMEN APPLICATION
Load at 1 to 2 mL/minute.

3. COLUMN RINSE
1 mL DI H2O; draw through under vacuum.
0.5 mL 0.01 M acetic acid; draw through under vacuum.
Dry column (4 minutes at 15 inches Hg).
50 uL CH3OH (no vacuum).
Dry column (1 minute at 15 inches Hg).

4. ELUTE ACIDIC AND NEUTRAL DRUGS (FRACTION A)
4 mL acetone:chloroform (50/50); draw through slowly under low vacuum (1 inch
Hg).

5. ELUTE BASIC DRUGS (FRACTION B)
2 mL EtOAc/NH4OH (98/2); use no vacuum.

section 3 - ANALYSIS

Add 100 uL of a 200 ug/mL Prazepam solution (internal standard). Mix/vortex. Evaporate each fraction to 100 uL at 40°C under N2. Inject 1 to 2 uL of each fraction into the gas chromatograph (GC).

CERTIFY GENERAL DRUG SCREEN ANALYSIS*
The following compounds have been extracted from urine and plasma samples using Certify extraction columns:
FRACTION (A): ACIDIC AND NEUTRAL DRUGS
Butalbital Clonazepam Methaqualone
Heptabarbital Diazepam Meprobamate
Hexobarbital Lorazepam
Metharbital Nitrazepam
Pentobarbital Oxazepam
Probarbital
Secobarbital

FRACTION (B): BASIC DRUGS
Amphetamine Levallorphan Procaine
Cocaine Mepivacaine Promethazine
Codeine Methamphetamine Trimipramine
Imipramine Morphine**
For more general information on these and other drugs of abuse, see John Wilson's "Abused
Drugs, A Laboratory Pocket Guide"; AACC Press; Washington, D.C.: 1990.
* Adapted from Chen, X.-H. et al. Journal of Forensic Sciences 1992, 37(1), 61-71.
** Requires 2 x 2 mL EtOAc/NH4OH (98/2) elution aliquots.
I don't perform these kind of analyses but changing a liner every batch of 20-30 samples is very common in our lab. It is the job of the liner to protect your system from whatever is not meant to be in the system by trapping it. I have sporadically -and jokingly- referred to liners as the condoms of GC.

The easiest thing you can do to make the liner less dirty is injecting less (or injecting diluted extracts). This requires of course the ability of your system to detect lower amounts.
We run dirty environmental samples and sometimes we can inject 50 samples and the liner is clean, sometimes 5 samples and the liner is black. With a matrix such as urine I can see the liner needing to be changed as often as you are changing yours.

We often have to change the gold seal at the bottom of the inlet when we change the liner to prevent analyte breakdown so don't forget to check that also. If there are a lot of high boiling point compounds present you will also want to check the split vent line, the copper tube running from the inlet to the split vent valve as those will become clogged and cause problems also. They can be rinsed with solvent or simply replaced with another length of copper tubing, and surprisingly you will see a reduction in analyte breakdown when changing that line.
The past is there to guide us into the future, not to dwell in.
Dear James_Ball and Rndirk,

Thank you so much for the answers.
Unfortunately we have to keep the sample concentrated in order to reach the desired LOD.

Please, let me know if there is other useful maintenances for dirty samples.

Best,

Johan :)
After reconstitution, does everything dissolves nicely? Having particles in the solution to inject is something you want to avoid at all cost in any chromatographic system.
Yes,
After reconstitution, I take care of centrifuging and only pipetting the supernatent.

Basic fractions are crystal clear, but the acidic fraction is showing a dark violet color (but looks transparent, without any visible particle).

I tried to filtrate it (acidic fraction) or to extensively wash it during the SPE but this color won't disappear (and I have to inject this fraction, which can contain drugs).
Is it an idea to create a SIM method for the compounds you want to detect instead of a scan method? This should increase your sensitivity, allowing you to inject less / dilute.

You can most likely find the ions to monitor for each of your compounds online.
SIM isn't an option for the moment because the main purpose is to detect as many drugs as possible.

It would be theoritically possible to use a SIM/SCAN method with a less concentrated sample but the drugs, which are not included in SIM wouldn't be detected in SCAN if the sample is too diluted. The main goal is to have a lower LOD than immunoassays.

Moreover, I was able to easily automize the interpretation in SCAN.
We encounter the same problem with required LOD so we just had to increase the maintenance schedule to compensate for the dirty samples. We also add a guard column of about 1m and each day before we begin analysis we remove 5-10cm of the guard column and exchange the inlet liner. If we see breakdown we either have to remove more guard column, or if severe we have to remove about 30-50cm of analytical column and install a new guard column.

Columns don't last forever and the more dirty the samples the faster they need to be replaced.
The past is there to guide us into the future, not to dwell in.
Thank you,
Can I ask you the model of your guard column?
I use the Intermediate Polarity Deactivated Guard Column 0.25mmID x 5m long Restek cat# 10043

It will depend on the solvent you are using though. The Intermediate is good for mid polarity to polar solvents.They make non-polar, polar, intermediate polarity, base deactivated, and a few specialty deactivation layers along with a non-deactivated one. You want to match the polarity of the deactivation to the injection solvent. IP is good for when we use both Ethyl Acetate (mid polar) and Dichloromethand (non-polar) solvents on the same instrument. For methanol or water injections you want the polar type.

We use these press fit connectors to join the column and guard column

https://www.restek.com/catalog/view/46605

If you have trouble sealing the connection you can also use some of the polyimide resin( https://www.restek.com/catalog/view/325 ) to help seal it, just put a thin layer on a paper wipe and wrap around the column and pull off the end of the column to coat about 1cm of the end of the column and guard before inserting it into the connector. This will give a very thin film of resin on the columns to enhance the seal. You also want to set up a curing method that keeps the inlet pressure about 5psi and ramps the temperature from 50c with a 5 minute hold up to the maximum temp of the column at about 5c per minute to slowly cure the resin in the connector, after that it will be sealed until you cut it off.

Don't use the method of putting a small bead of resin on the outer end of the connector, I never had good luck with that method.
The past is there to guide us into the future, not to dwell in.
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