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2008 vol. 1
Clinical/Forensics
Introduction
Analyzing amphetamines by GC/MS is challenging whether the compounds
are derivatized or underivatized. Underivatized amphetamines appear as
irregular and asymmetric peaks, which are difficult to integrate, and may lead
to irreproducible results. Derivatized amphetamines result in symmetric
peaks, but derivatizing reagents can contaminate the inlet/column. This con-
tamination can shorten column lifetime and cause noisy, elevated baselines that
interfere with the analysis of target compounds.
In this study, we evaluated the effects of several sample pretreatment methods.
These methods included: 1) no pretreatment, 2) converting the salt forms into
free bases, 3) derivatizing the free bases with heptafluorobutyric acid anhydride
(HFAA), and 4) derivatizing the free bases with HFAA followed by a clean up.
Our objectives were to obtain symmetric shapes, reduce baseline noise, and
maintain low column bleed from injection to injection for GC/MS analysis.
Procedure
The first method had no pretreatment. The untreated standard was prepared
in methanol and diluted to a final concentration of 100µg/mL. It was then
injected without any further preparation. The second pretreatment involved
converting the drug standard to the free base form. The free base forms were
prepared by mixing the standard (100µg/mL) with water, then adding satu-
rated sodium borate water, and extracting the amphetamines with butylchlo-
ride. The resulting sample was then analyzed by GC.
The third pretreatment procedure included both conversion and derivatiza-
tion. The HFAA derivatized amphetamines were prepared by converting the
compounds to free bases (as described above), reacting with derivatizing
reagent HFAA, and diluting the sample before injection. The fourth pretreat-
ment procedure consisted of free base conversion, HFAA derivatization, and a
clean up step to remove the acidic byproducts of derivatization. The clean up
procedure included mixing the sample with a phosphate buffer (pH=7.0)
before dilution, removing the butylchloride layer, and then diluting the sam-
ple just before injection. An Rtx®-5MS column (30m x 0.25mm ID x 0.25um)
was used for analysis; instrument conditions are presented in Figure 1.
Repetitive GC/MS runs (over 190 injections) were evaluated to confirm sym-
metry, baseline, and bleed results.
Results
Analyzing untreated amphetamine and methamphetamine results in peak
doublets caused by the presence of both the salt (hydrochloride) and free base
forms (Figure 1). Peak doublets were eliminated by conversion to free base
form, however, some tailing was still observed (Figure 2). This pretreatment
improves reproducibility, but is still not optimal as tailing can cause irrepro-
ducible integration and significant variation in peak area counts.
The most symmetric peak shapes were obtained by derivatizing the ampheta-
mines with HFAA (Figure 3). Although peak shape was improved, the acidic
derivatization byproducts generated a noisy baseline and shortened column
life. This system contamination increases injector and column maintenance.
Accurate, Reproducible Amphetamines Analysis
Clean Up Procedure Improves Chromatography and Reduces Maintenance
By Kristi Sellers, Clinical/Forensic Innovations Chemist, and Amanda Rigdon, Innovations Chemist
• Derivatization improves peak symmetry, for more accurate results.
• Clean up procedure reduces system contamination, and extends column lifetime.
• Rtx®-5MS column produces a stable baseline for derivatized compounds, ideal for GC/MS analysis.
Figure 1
Untreated standard contains both
salt and free base forms causing inaccu-
rate, irreproducible results.
Figure 2
Conversion to free base form
improves chromatography, but produces
tailing factors over 2.0.
GC_PH00973
Column:
Rtx
®
-5MS, 30m, 0.25mm ID, 0.25µm (cat.# 12623)
Sample:
100µg/mL amphetamine and methamphetamine in
methanol
Inj.:
1µL, split (split ratio 10:1), 4mm single gooseneck
w/ wool inlet liner (cat.# 20798-211.1)
Inj. temp.:
250°C
Carrier gas:
hydrogen, constant flow
Flow rate:
1mL/min.
Oven temp.:
70°C to 250°C @ 15°C/min. (hold 5 min.)
Det:
FID @ 300°C
1. amphetamine, free base
2. amphetamine, salt
3. methamphetamine, free base
4. methamphetamine, salt
GC_PH00974
See Figure 1 for conditions.
1. amphetamine, free base
2. methamphetamine, free base