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CLINICAL/FORENSICS ARTICLE

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.

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 the derivatizing reagents can contaminate the inlet and column. This contamination can shorten column lifetime and cause noisy, elevated baselines that interfere with analyzing the 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

All preliminary pretreatment evaluations were performed using an Rtx®-5MS column (30m x 0.25mm ID x 0.25µm), a base deactivated inlet liner packed with IP deactivated wool, and a GC/FID. Standards containing amphetamine and methamphetamine were used for initial evaluations, and standards containing amphetamine, methamphetamine, MDA, MDMA, and MDEA were used for the final evaluations. Once a final pretreatment procedure was determined, repetitive pretreatments and GC/MS runs were made to confirm reproducibility for MS work.

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 method involved converting the drug standard to the free base form in order to improve peak shape. The free base forms were prepared by mixing the standard (100µg/mL) with water, then adding that solution to saturated sodium borate water, and extracting the amphetamines (free bases) out of the water solution with butylchloride. The resulting sample (in butylchloride) was then analyzed by GC.

The third pretreatment procedure included both conversion and derivatization. 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 into the GC. The fourth and final pretreatment 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 sample just before injection into the GC. Instrument conditions are presented in Figure 1. Following data acquisition, tailing factors were calculated, baselines were studied, and repetitive GC/MS runs (over 190 injections) were evaluated to confirm peak symmetry, clean baselines and low column bleed.

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). In this case, complete resolution of the two forms is apparent. In other cases, this can be seen as severe tailing or peak splitting. Peak doublets were eliminated by conversion to free base form, however, some tailing was observed (Figure 2). This pretreatment improves reproducibility, but is still not optimal as tailing can cause irreproducible integration from injection to injection, leading to large variations in area counts.

The most symmetric peak shapes were obtained by derivatizing the amphetamines with HFAA (Figure 3). Although peak shape was improved, retention times shifted due to the higher molecular weight produced, and acidic derivatization byproducts generated a noisy baseline. This pretreatment method resulted good peak shape and reproducible area counts (i.e. accurate integration), but the contamination from the derivatizing reagent will increase injector and column maintenance, and shorten column lifetime.

Incorporating a post conversion/derivatization clean-up procedure removed derivatization contaminants while maintaining chromatographic quality (Table 1), thus reducing the need for frequent system maintenance. As shown in Figure 4 the high background noise is reduced significantly while peak symmetry is maintained. These benefits were also seen when samples were analyzed by GC/MS (Figure 5).

Conclusion

Even though the conversion/derivatization method with the clean-up procedure is more time consuming for sample preparation, it produces symmetric peaks while reducing the amount of contamination that can enter the GC system. This method will ensure accurate, reproducible area counts, a clean GC column and system, and a stable baseline, even for GC/MS work.

Figure 1: The presence of both salt and free base forms causes inaccurate integration and irreproducible area counts.
  1. amphetamine, free base
  2. amphetamine, salt
  3. methamphetamine, free base
  4. methamphetamine, salt

Column:

Rtx®-5MS, 30m, 0.25mm, 0.25µm (cat.# 12623)

Sample:

100µg/mL amphetamine and methamphetamine in methanol

Inj.:

1.0µL 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 (hold 1 min.) to 250°C @ 15°C/min. (hold 5 min.)
Det.:
FID @ 300°C

GC_PH00973
Figure 2: Conversion to free base form improves chromatography, but produces tailing factors over 2.0.
  1. amphetamine, free base
  2. methamphetamine, free base

See Figure 1 for condtions.


GC_PH00974
Figure 3: Derivatizing with HFAA yields symmetric peaks but results in system contamination and a noisy baseline.
  1. amphetamine
  2. methamphetamine

See Figure 1 for condtions.


GC_PH00975
Figure 4: A post-derivatization clean up procedure results in symmetric peaks and a clean baseline.
  1. amphetamine
  2. methamphetamine
  3. MDA
  4. MDMA
  5. MDEA

See Figure 1 for condtions.


GC_PH00976
Figure 5: Post-derivatization clean up also produces symmetric peaks and a stable baseline when analyzed by GC/MS.
Peak List: Tailing Factor:
1. amphetamine 1.109
2. methamphetamine 0.992
3. MDA 1.106
4. MDMA 1.068
5. MDEA 1.113

Column:

Rtx®-5MS, 30m, 0.25mm, 0.25µm (cat.# 12623)

Sample:

100µg/mL each amphetamine, methamphetamine, MDA, MDMA, and MDEA extracted from methanol and HFAA derivatized

Inj.:

1.0µL splitless (hold 0.5 min.), 3.5mm custom splitless inlet liner w/IP deacitvated wool;
Inj. temp.:

220°C
Carrier gas:

helium, constant flow

Flow rate:

1.25mL/min.
Oven temp.:

70°C (hold 1 min.) to 290°C @ 15°C/min. (hold 4 min.)
Det.:
MS
Transfer line temp.:
280°C
Scan range:
43-450amu
Ionization:
EI
Mode:
scan

GC_PH00972

Table 1: Tailing factor comparison of pretreatments.

Pretreatment

TF Amp

TF Meth

TF MDA

TF MDMA

TF MDEA
Sodium Borate Wash (GC/FID) 2.115 2.837 NA NA NA
HFAA Only (GC/FID) 1.010 0.989 NA NA NA
HFAA w/Post clean Up (GC/FID) 0.981 0.996 1.007 0.997 0.992

NOTE: A perfectly symmetric peak exhibits a tailing factor of 1.0. Tailing factors shown were generated using the USP tailing factor calculation.