Wash kinetics

For the accurate interpretation of a positive hair analysis result, it is necessary to subject hair to specially developed wash and extraction procedures. During the development of these procedures, we discovered several important properties of hair, properties which were essential for defining three unique wash kinetic criteria and their cutoff levels which, in conjunction with washing, enhance the acciuacy of the interpretation of a positive hair analysis result. Additional certainty can be achieved by the measurement of metabolites or metabolite/drug ratios, by the development of ultrasensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) or ion... 

Wash kinetics of contaminated hair and hair from a drug user. Hair treatments , dry isopropanol washes , phosphate buffer , enzymatic digestion. 

There is considerable variation in how fast different hair specimens approach plateau conditions in their wash-out kinetics. Most nonporous xmcontaminated hair attains plateau conditions within 1.5 h heavily contaminated and porous hair specimens may require 3 h or longer. This variabihty in the wash kinetics of different hair specimens has to be addressed under mass production conditions where individualized monitoring of the attainment of plateau conditions cannot be performed for each hair specimen. 

Under mass production conditions, all hair samples are washed in an identical manner by the truncated wash procedure. This consists of one 15-min wash with dry isopropanol and three 1/2-h washes with phosphate buffer. Not aU hair samples will have reached plateau conditions by this procedure. To identify those samples which require further washing, we have defined two additional wash kinetic criteria " the curvature ratio, R, and the extended wash ratio, R w These are defined as follows ... 

Rew/ on the other hand, is the worst-case estimate of the number of 1/2-h aqueous washes it would have taken to wash out the residual drug found in the hair digest if the residue had been contained in the semiaccessible domain. It is the worst-case estimate because the slope of the wash kinetics operating during the third wash is assumed to remain constant — a highly unlikely situation under nonplateau conditions. Rew values become very large numbers which, in the limit, approach infinity as perfect plateau conditions are attained. 

Rew and R ratios were used in conjunction with the truncated safety zone ratio, Rj z, to describe the results obtained with populations of known drug users. The percentage distributions of values for these wash kinetic parameters are given in Tables 2, 3, and 4. Based on the data from these populations of known drug users and the wash kinetic values obtained with artificially contaminated hair, the following cutoff levels were set for the truncated wash procedure R< = 1.3, Rew = 10/ Rtsz = 0.33. Samples are judged to be contaminated if any one of the three wash kinetic parameters falls below its cutoff value. 

As with urinalysis cutoffs, the further the wash kinetic results are from their cutoff values, the more certain is a finding of drug use. In general, our data showed that the values of the wash kinetic parameters of porous hair samples tended to cluster in regions close to the cutoff levels whereas those of nonporous hair tend to be further... 

Wash kinetics of 6-monoacetylmorphine-contaminated hair. O = porous hair. 

These results are in striking contrast to those obtained with the hair from heroin users (Figure 3). Essentially perfect plateau wash kinetics were obtained for 6-MAM, morphine, and codeine. Most important, the amounts of 6-MAM released from the inaccessible domain by digestion easily meets the R sz criterion of heroin use. It should also be mentioned that had the alcohol washes been continued, then the alcohol wash kinetics would have been similar to curve AB in Figure 1. 

Wash kinetic/digestion profile of 6-monoacetylmorphine ( ), morphine (A), and codeine (O) in the hair of a heroin user. All samples received an initial wash with dry ethanol ( ). This was followed by washes with phosphate buffer (pH 5.5) and release of remaining drugs by enzymatic digestion. 

Several recent papers by Blank and Kidwell have questioned the reliability of our wash kinetic procedures. Their criticism, which in many instances is marred by the faulty application of our technology, has demonstrated the need for further clarification of the rationale upon which our procedures, particularly the wash kinetic procedures, are based. 

Another aspect of our diagnostic algorithm that is not sufficiently appreciated is that the validity of the extended wash ratio and the safety zone ratio depends on the wash kinetics showing a significant amount of curvature as measured by the... 

In a recent review. Blank and KidweU have questioned the adequacy of the wash procedures used in various hair analysis laboratories. In their review they also extended their earlier criticism23-24 of our wash kinetic approach. 

In light of the obvious flaws in their evaluation, we once again contend that their experiments did not constitute a valid evaluation of om" wash kinetic procedures. Nor do we agree with them that their described isotope procedure is suitable for contamination/decontamination studies. As a matter of fact, it is likely that an inherent difficulty in their isotope procedures, which we shall describe shortly, may have created the observed difference between their first and second studies. It is also likely that insufficient attention was given to critical elements in their contamination/decontamination experiments. Since we have not described the latter in our previous publications, we will briefly simunarize these now and then comment on a general problem with their isotope method. 

In our experiments, hair (20 mg) is soaked in 2 mL of phosphate buffer (pH 5.5) containing cocaine hydrochloride (1,000-10,000 ng/mL) for 1 h at 37°C. To avoid interference from a variety of factors (see below) including possible adsorption of drugs on the walls of the polystyrene test tubes, the contaminated hair is subjected to the following treatments before commencing with the wash kinetic experiments in a new test tube. 

Even though the majority of results were below the adult endogenous cutoff level (not to mention the tenfold higher cutoff level for children) and in spite of the presence of benzoylecgonine which indicated passive exposure. Smith and KidwelPi drew the erroneous conclusion that our wash kinetic criteria failed to... 

This has been our experience with the challenging task of differentiating between the hair of marijuana users and contaminated hair from nonusers who were only exposed to marijuana smoke. Here only the measurement of the metabolite, carboxy-THC, but none of the wash procedures or wash kinetic approaches that we have successfully applied to the other drugs have led to satisfactory results. Our ability to measure digoxin in small hair samples, even though this was taken in doses as... 

The rate of water oxidation which resulted specifically from the activation of Ca or Sr " " was determined by subtracting the residual rate of O2 evolution which remained after the salt wash. Kinetic constants for the activation of water oxidation by Ca " " and Sr were determined by the HYPERO program of Cleland (16) which was translated into BASIC as described (11). 

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