Introduction

Methaqualone

Methaqualone is a sedative that is frequently misused with alcohol. If its concentration is too high, it will react with alcohol and cause problems like unconsciousness and death. How can we determine the presence and concentration of methaqualone?


The authors' method is a gas-liquid chromatograph of methaqualone in a serum solvent. Their method is fast and robust towards biological substances.    


If the concentration of methaqualone is too high, it will react with alcohol and will bring about hazardous effects to the body for example unconsciousness or death.

Overdose concentrations of Methaqualone generally range from 2.0
mg/liter to 22 mg/liter.
A therapeutic dosage of over 8mg/liter results in unconsciouness.

Materials

Reagents

Methaqualone Solvent - serum

Internal standard(PBCA) solvent - Chloroform

Alkaline buffer - Borate buffer (pH 10.5) (Aqueous Phase) - This buffer is prepared by
adding about 8ml of NaOH(6 mol/liter) to 100 ml of a saturated sodium borate solution which will yield a pH 10.5 borate buffer.


Why do we need this pH 10.5 buffer?

PBCA and methaqualone functioned best between pH 9.5-11.0. Also interferences from biological substances were less. Therefore, the authors decided to use a pH of 10.5.



Internal Standard: PBCA( N-Propionyl-2- benzoyl-4-chloro-aniline)

Analyte: Methaqualone (prepared from the pure standard of Methaqualone hydrochloride)

Concentrations of
5, 10, 20, 30, and 40 mg/liter were prepared <------------------------ These are the Standards

The serum standards were placed into individual 5-ml
vials, frozen, and stored at 4 degrees C until needed.

Apparatus:
F&M 402 gas chromatograph
dual hydrogen-flame detectors(FID)
A U-shaped glass column (150 cm, 2 mm i.d.)
was packed with “3% OV-1 (methyl silicone) on
100/120 mesh Gas Chrom Q”
flow rate for helium, hydrogen, and air were
30, 20, and 380 ml/min,
column temperature was 210  degree C; detector temperature was 300
degree C; flash-heater temperature was 310 degree c.
electrometer settings were: range X10 and attenuation X4. Strip-chart recorder (Hewlett-Packard
Model 7127A) speed was 1.27 cm/mm.

Procedure

Preparation of solution to be injected into gas chromatograph:
Put 2.0ml serum, 1.5ml of pH10.5 borate buffer, and 0.50ml of PBCA dissolved in chloroform into a 15-ml glass-stoppered centrifuge tube pipet.
Vortex the contents for 3.5min. Centrifuge the tube for 5min at 2500rpm. Discard the aqueous layer. Using a 10 microliter syringe, inject 4microliters of the organic layer into the gas chromatograph.

Figure 3 shows a typical chromatogram of a sample containing PBCA, methaqualone and serum.

Figure 4 illustrates the chromatogram of sample containing only serum. This shows that the serum is pure and is free of interferences.

Figure 5 is a chromatogram of a sample containing methaqualone, from a patient using the authors' method.

Calibration Graph: Ratio of peak area methaqualone/peak area of PBCA( y-axis) against conc. of methaqualone v/v (x axis)

This calibration graph is plotted from results of the methaqualone standards of 5,10,20,30,40 mg/liter concentrations prepared previously.

Patient's sample of unknown concentration of methaqualone is put into the GC using the author's method. Peak area of methaqualone/peak area of PBCA(Internal standard) of sample is obtained. Concentration of methaqualone in patient is determined by using the calibration graph of standards. 

Results

1) Precision - assessed the precision of both the instrument(GC) and their method.

Instrumental precision: They injected 5, 10, and 20 mg/liter extracts of methaqualone, 10
times each. The %RSD ranged from 0.4% to 0.9%.

Method precision: Injecting a 10 mg/liter methaqualone standard( in serum) 23 times. The Standard deviation was 0.16mg/liter, with a %RSD of 1.6%.  
They also assessed day-to-day precision by injecting the same sample on 20 different days. The standard deviation was 0.5mg/liter, with a %RSD of 5.6%.


2) Stability

methaqualone in serum did not deteriorate when stored in room, refridgerator, freezer for a year.
PBCA in chloroform did not deteriorate when stored in room temp for a year.
both are relatively stable, this will not affect experimental readings.



3) Linearity and Sensitivity

linear response was found when 5-40mg/L of methaqualone in serum was used in FID(flame ionization detector)


4) Interference


They did a test to determine if any commonly used drugs that could be present in the serum would interfere with the credibility of the test.
They performed GLC with the drugs in the presence of methaqualone and the internal standard PBCA to see if these drugs affect the peak areas or symmetry of the methaqualone/PBCA peaks.
None of them showed any interference. However,they did not test the metabolites of the drugs.

Note: pentazocine, one of the drugs tested elutes close to PBCA, giving a tailing hump to the PBCA peak.
Under normal conditions, the maximum dosage of pentazocine is 0.16mg/liter which is too small to interfere with the experiment.

Discussion

The authors found that it was necessary to synthesize an internal standard-PBCA as it gives the best results.
Why?
1) drugs cant be used as internal standards because of self interefence.(they contain methaqualone)
2) non drug compounds eluted in the same position as methaqualone.
3) other compounds had retention times that were too long or too short. Retention times can affect the resolution and symmetry of the peaks.


Confirming peak signals
They performed their method on a column packing containing a different liquid stationary phase. (3% OV-17)
3% OV-17 on 80/100 mesh Chromosorb W (HP) packing can be used to confirm positive results for the 3% OV-1 packing.
(which is what their method is using as a liquid phase)

Note: Figure 3 is a chromatogram of methaqualone in serum using their method and a 3% OV-1 packing.
Figure 7 is a chromatogram of methaqualone in serum using their method but a 3% OV-17 packing.

Why is OV-17 not used as a liquid phase for their method?

OV-17 is used for this test and not for the method because
1) increased time needed to elute the sample.
2) higher chance of interference from other drugs.
3) peaks eluting very near or in the same area as methaqualone or the internal standard.


Conclusion

This method is reliable, time-saving and can be done by someone with little GC experience.
Also, it can quantify the amount of methaqualone in a patient within 15 minutes after receiving the patient sample.

References

BLTC (2012, January). M e t h a q u a l o n e. THE GOOD DRUG GUIDE. Retrieved July 5, 2012, from http://biopsychiatry.com/methaqualone/index.html

Evenson, M. A., & Lensmeyer, G. L. (1973). Qualitative and Quantitative Determination of Methaqualone in Serum by Gas Chromatography. CLINICAL CHEMISTRY (1974),20(2), 249-254. doi:Dec 2, 1973 Retrieved from http://www.clinchem.org/content/20/2/249.full.pdf

Qualitative and Quantitative Determination of Methaqualone in Serum by Gas Chromatography.


Newspaper.li (n.d.). Chemistry . Newspaper.li . Retrieved July 23, 2012, from http://http://newspaper.li/chemistry/