a p p l i c at i o n N o t e

Gas Chromatography/
Mass Spectrometry

Author
Timothy D. Ruppel
PerkinElmer, Inc.
Shelton, CT 06484 USA

Opiates in Urine by Introduction

The United States Department of Health and Human Services
SAMHSA GC/MS (DHHS), Substance Abuse and Mental Health Services Administration
(SAMHSA) regulates urine drug testing programs in the mandatory
guidelines for the Federal Workplace Drug Testing Program. These
Mandatory Guidelines require a laboratory to conduct two analytical
tests before a urine specimen can be reported positive for a drug, the
initial drug test and the confirmatory drug test. The initial drug test
is performed by immunoassay screening for the five drug classes
(i.e., amphetamines, cocaine, opiates, phencyclidine, and marijuana).
Examples of immunoassay screening would include radioimmunoassay
(RIA), enzyme immunoassay (EIA, EMIT) or others.

Samples found positive to the immunoassay screening are subjected

to a second confirmatory test by chromatographic separation and
identification by mass spectrometry. SAMHSA defines the method
quantification cutoff level as 2000 ng/mL each for codeine and morphine.
If morphine is detected above 2000 ng/mL, then an additional
quantification for 6-acetylmorphine is suggested. 6-AM is a unique
metabolite indicating the use of heroin. 6-AM cutoff level is 10 ng/mL.
 The general procedure for drug confirmatory test in urine Instrumentation
follows the 7 steps listed below: Gas Chromatograph: PerkinElmer Clarus 680 GC
1. Add a deuterated internal standard to the urine. Injector: Capillary injector using pressure pulsed splitless
2. Adjust urine pH. injection, 250 °C.
3. Hydrolyze urine (opiates and cannabinoids only). Injection port liner: Siltek™ with wool (Cat No. N6502010).
4. Extract drugs from urine using solid phase extraction
(SPE), evaporate to dryness. GC Column: Elite-5 (5%Phenyl/95% Methyl Silicone) –
12 m x .200 mm x 0.33 µm (Cat No. N9316110).
5. Derivitize the extract (except for PCP), evaporate to dryness.
6. Reconstitute extract into organic solvent. Helium carrier – 2 mL/min

7. Inject 1-3 µL into gas chromatograph/mass spectrometer GC oven: Start temperature 100 °C hold for 0.5 min, then
for identification and quantitation using three ion ratio 20 °C/min to 310 °C hold 4 min.
reporting software.
Pressure pulsed, splitless injection
Glassware
This procedure raises the injector pressure during the injec-
All glassware, including autosampler vials and low volume
tion process to put more sample onto the column in a
vial inserts must be silanized to prevent adsorption of sample.
narrow band and then reduces the carrier gas to normal
Soak all glassware in 10% DMDCS/Toluene for 10 min. operational linear gas velocity for chromatography. This is
Rinse in methanol, rinse in hexane, air dry. accomplished with timed events such as the following:

CAR2 set to 5 mL/min at -0.71 min (raise pressure before

Reagents list
injection).
Beta-glucuronidase
SPL2 set to 0 at -0.70 min (splitless injection).
Sodium Hydroxide
CAR2 set to 2 mL/min at 0.7 min (operating flow after
Hydrochloric Acid (conc.) injection).

Acetic Acid, 100 mM = 2.86 mL glacial acetic acid diluted to SPL2 set to 50 at 0.8 min (open split vent after injection).
500 mL DI water
Mass Spectrometer PerkinElmer SQ8 MS, 270 L/sec
Phosphate buffer, 100 mM pH6 = 1.7 g Na2HPO4 + 12.14 g turbomolecular pump, EI mode.
Na2PO4 dilute to 1000 mL with DI water.
All data is collected in selected ion monitoring mode (SIM)
Adjust to pH6 with 100 mM Na2HPO4 (raises pH) or acquiring 20-30 msec per ion.
100 mM Na2HPO4 (lowers pH).
A primary ion is used for identification and quantitation
Methylene Chloride/Isopropanol/Ammonium Hydroxide while 2 additional ions are used for confirmation of
(78:20:2) extraction solvent = 40 mL IP-OH + 4 mL identification.
con NH4OH + 156 mL MeCl2. Make fresh daily.
Three ion ratio chromatograms must all apex within ±2
Drug standards and deuterated internal standards are available scans of standard retention time. Ion ratios must fall within
from Cerillant® (Round Rock, TX). ±20% of standard ratios. Deuterated internal standards may
use only 2 ions, a primary ion and only 1 confirmation ion.
Internal standard: d3-codeine, d3-morphine, d3-6-acetylmor-
phine.

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PFPA SIM ions: Codeine: 282,445, 446 d3-Codeine: 285, 448 RT: 7.16 min
Morphine: 414, 430, 577 d3-morphine: 417, 580 RT: 7.35 min
6-acetylmorphine: 414, 361, 473 d3-6-acetylmorphine: 417, 479
BSTFA SIM ions: Codeine: 371, 234, 343 d3-Codeine: 374, 237
Morphine: 429, 287, 324 d3-Morphine: 432, 290

Solid Phase Extraction Experimental

Drugs are extracted from the urine sample matrix by solid Extraction Procedure: 3 mL urine + ISTD. Hydrolyze sample
phase extraction (SPE) with a polymeric resin cartridge. The to break glucuronide bonding.
drugs are retained as the urine is passed through the resin
bed. Washing the bed can remove salts and other contami- SPE column extract: Condition column with 3 mL methanol,
nants. Eluting the drugs off the resin bed with a stronger then 3 mL DI water, then 1 mL 100 mM phosphate buffer
solvent completes the cleanup process from the urine. pH6.
Extraction cartridges used were Supra-Clean SPE Columns
Extract sample, wash column with 3 mL DI water, then 1 mL
C18-S 200 mg/3 mL 50 u (Cat No. N9306462).
100 mM Acetic Acid, then 1 mL methanol.

Hydrolysis Elute column with 3 mL Methylene chloride:Isopropanol:

Hydrolysis is necessary in some drugs to remove glucuronide Ammonium Hydroxide (78:20:2) into conical tube.
bonding which prevents the solubility and extraction of the
Evaporate to dryness <50 °C. Derivitize with 50 uL PFPA,
drug. This preparation before extraction is usually accom-
add 25 uL PFPOH. Cover with N2, cap, mix, heat 70 °C
plished either by enzyme or acid hydrolysis of the sample.
(20 min).
Both procedures are outlined below.
Evaporate to dryness <50 °C.
Enzyme Hydrolysis Procedure
Reconstitute in 100 µL ethyl acetate, transfer to low volume
Combine 3-5 mL urine sample with ISTD and 2 mL beta-
autosampler vial insert, inject 1 µL.
glucuronidase, vortex, heat 3 hours at 65 °C.

Cool, centrifuge, decant and keep top layer. Adjust pH to Alternative Derivitization Procedure
6.0 with 700 uL 1.0 N NaOH. Reconstitute dried extract with 50 uL ethyl acetate, add
50 uL BSTFA with 1% TMCS.
Acid Hydrolysis
Cover with N2, cap, mix, heat 70 °C (20 min), cool, do not
Combine 3-5 mL urine sample with ISTD and 500 uL
evaporate, inject BSTFA solution directly.
concentrated HCl, vortex, heat 30 min at 120 °C.

Cool, centrifuge, decant and keep top layer. Add 1 mL Calibration Range
7.4 N NH4OH, vortex. Adjust pH to 6.0 with 1-3 mL of 10% cutoff (200 ng/mL), 40% cutoff (800 ng/mL), 100%
500 mM phosphoric acid. cutoff (2000 g/mL), 125% cutoff (2500 ng/mL), 500%
cutoff (10000 ng/mL), 1000% cutoff (20000 ng/mL)

3
 Results

Limit of Quantitation: 20 ng/mL from 1mL urine.

Limit of Detection: < 2 ng/mL from 1 mL urine.
Linear Correlation coefficient (R2) >0.999 20 ng/mL – 20000 ng/mL.

Results for 6-acetylmorphine PFP

4
Conclusion
The GC/MS analysis of codeine and morphine in this applica-
tion has demonstrated the limit of quantitation at or below
20 ng/mL in urine a 10 fold factor lower than the limit of
quantitation requirements of the Federal Workplace Drug
Testing Program. Forensic and clinical laboratories can use
the same method for toxicology samples in non-regulated
drug testing. Fast sample throughput was increased through
the use of a short GC column, fast flow rate into the mass
spectrometer, very fast cooling GC oven and autosampler
pre-rinsing options.

The PerkinElmer SQ8 GC/MS system operating in SIM mode

provided the sensitivity and spectral data necessary to
generate legally defensible results. The TurboMass GC/MS
software includes the reporting capability required to An example of a customizable three-ion ratio report.
present three-ion-ratio data in a format that is simple
and easy to understand. References
1. Disposition of Toxic Drugs and Chemicals in Man, 8th Ed,
Randall C. Baselt, Biomedical Publications, 2008.

2. Mandatory Guidelines for Federal Workplace Drug Testing

Programs, Fed Reg, 73: 71857 (Nov 25, 2008).

3. Mandatory Guidelines for Federal Workplace Drug Testing

Programs, Fed Reg, 75: 22809 (April 30, 2010).

4. Pierce Catalog (Rockford, IL).

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