Drugs of Abuse in Urine

Automatic Sample Preparation Prior to GC/MS Analysis

Testing of urine specimens under the guidelines of the U.S. Department of Health and Human Services (DHHS) requires initial testing by an immunoassay technique, followed by confirmation of all presumptive positive specimens by gas chromatography/mass spectrometry (GC/MS).^1,2 (Federal Register, 1988; National Laboratory Certification Program, 1994)

The sample preparation necessary in the confirmation assays is complex, requiring skilled personnel who must attentively and precisely perform a series of manual procedures on batches of specimens, calibrators, controls and blanks over a period of four to five hours.

Current Analytical Approaches
In general, somewhat simple initial specimen manipulation (adding internal standards and buffering the specimens) is followed by solid­phase extraction (SPE) to isolate the analytes of interest from the rest of the matrix, and then by derivatization to form more volatile compounds amenable to GC/MS analysis. For opiates, initial specimen preparation is more complex; for PCP (phencyclidine), specimen preparation is simpler, because derivatization for increased volatility is unnecessary.

Benefits of PrepStation Automation
The HP PrepStation allows a chemist to automate most sample preparation steps encountered in confirmation assays. Hands­on labor is significantly reduced, often by a factor of up to four. With a system comprised of a PrepStation integrated with a gas chromatograph and a mass selective detector, there is no user intervention between inputting batches of vials containing specimens, calibrators, controls, and blanks and receiving the analytical reports. SPE and derivatization are handled automatically.

As a result, laboratory personnel are free to do other tasks.

Validating PrepStation Performance
Confirmatory methods of forensic laboratories must be performed with adequate quality assurance to provide results that are legally and scientifically defensible.³ (B.A. Goldberger and M.A. Huestis) All laboratories must maintain documented standard operating procedures for each assay and must monitor the quality of laboratory data. When an assay is modified, the new method must be validated and demonstrate acceptable performance. Depending upon the significance of the modification, assay validation studies may include:

• Assay characterization
• Accuracy and precision studies
• A parallel study with authentic specimens
• A potential carryover study

Recently, a series of studies validated HP PrepStation performance for various drugs of abuse in urine: benzoylecgonine,⁴ THC­acid,⁵ opiates,⁶ and PCP.⁷ (B.A. Goldberger and W.S. Miles)

The overall goal of these studies was to demonstrate increased laboratory productivity by automating the majority of manual sample preparation steps used in confirmatory assays while meeting validation requirements for each class of compound. This goal was met.

Highlights of the Results
In all the validation studies, hands­on labor was reduced by at least a factor of four by automating most sample preparation steps. Compared to manual methods, accuracy was comparable and within­run and between­run precision was improved. In the benzoylecgonine study, intra­run accuracy and precision were evaluated, resulting in a mean of 179.33 ng/mL and a coefficient of variance of 0.91 percent for 10 samples with benzoylecgonine at a target concentration of 177 ng/mL.

Summary of the PrepStation Validation Studies
Components	Assay Characterization		Between Run: Overall % Coefficient of Variance		Parallel Studies: Authentic Specimens	Potential Carryover
	Limit of Quantitation	Linearity	Low Control	High Control	Correlation Coefficient
Benzoylec­ gonine	30 ng/mL	30-1000 ng/mL	3.42%	2.00%	0.998	None
THC-Acid	6 ng/mL	6-100 ng/mL	5.30%	2.12%	0.998	Insignificant*
Opiates	120 ng/mL	120-1000 ng/mL
Codeine			1.51%	1.90%	0.999	None
Morphine			2.45%	2.32%	0.972	None
PCP	10 ng/mL	10-200 ng/mL	2.04%	1.89%	0.999	Insignificant*

*Carryover was insignificant (< 1%) following unrealistically high concentrations.

References
1. Mandatory Guidelines for Federal Workplace Drug Testing Programs; Final Guidelines; Notice, Federal Register, 53, p. 11970 (1988).

2. Guidance Document for Laboratories and Inspectors, U.S. Department of Health and Human Services, National Laboratory Certification Program, revised August 29, 1994.

3. B.A. Goldberger and M.A. Huestis. GC/MS: Quality Assurance/Quality Control Procedures for Forensic Urine Drug Testing Laboratories. HP Publication No. 5963­9743E (1995).

4. B.A. Goldberger and W.S. Miles. Benzoylecgonine in Urine: Validating a PrepStation/GC/MSD System that Integrates Automated Sample Preparation with Analysis. HP Publication No. 5964­3705E (1995).

5. B.A. Goldberger and W.S. Miles. HC­Acid in Urine: Validating a PrepStation/GC/MSD System that Integrates Automated Sample Preparation with Analysis. HP Publication No. 5964­5950E (1995).

6. B.A. Goldberger and W.S. Miles. Opiates in Urine: Validating a PrepStation/GC/MSD System that Integrates Automated Sample Preparation with Analysis. HP Publication No. 5964­5948E (1995).

7. B.A. Goldberger and W.S. Miles. PCP in Urine: Validating a PrepStation/GC/MSD System that Integrates Automated Sample Preparation with Analysis. HP Publication No. 5964­5949E (1995).