CA2133560C - Hair analysis method - Google Patents

Abstract

A method for the direct analysis of analyte indicative of marijuana exposure found in keratinized structures, e.g., hair, fingernails and toenails, which comprises preparing a mixture containing dithiothreitol or dithioerythritol, a protease suitable for the digestion of the keratin structure and a sample of the keratin structure; permitting the enzyme to at least substantially digest the sample of keratin structure to form a digest solution, followed by mixing the digest solution with a suspension of an ion exchange resin to remove an interfering, cross reacting substance naturally found in hair and finally subjecting the digest solution to analysis to determine the identity and amount of marijuana analyte in the keratin structure sample. To accelerate the method, cupric sulfate may be added to the mixture after degradation of the keratin sample in order to deactivate the activator. The enzyme may be a protease with papain, chymopapain, and proteinase K being preferred for use in the invention. Exemplary ion exchange resins useful in the method according to the invention are DEAE Sephadex (Diethylaminoethyl Sephadex) and DEAE Sepharose (Diethylaminoethyl Sepharose).

Description

HAIR ANALYSIS METHOD
FIELD OF THE INVENTION
This invention relates to an improved analytical method which effectuates the relatively rapid solubilization of hair and direct analysis of organic analytes, e.g., drugs of abuse, present in hair and other keratinized structures, e.g., fingernails and toenails, without effecting the structure of the analyte or being detrimental to biological analyte probes, e.g., antibody, RNA/DNA and bio-receptor probes. The analyte can be analyzed by adding the analyte probe directly to the solubilized keratin structure containing the analyte to determine the identity of the analyte as well as the extent and duration of its consumption by a subject.

HACXGROUND OF THE INVy'NTION
In the past, hair analysis technigues for the detection of trace metals were developed that purported to provide information on an individual's nutritional status. One objection to the use of these techniques is the difficulty of distinguishing between trace metals deposited in hair from the bloodstream and metals which have become embedded in hair through external contact with, for example, water and cosmetic agents. Consequently, these techniques are not considered useful by the medical community for diagnosing nutritional problems, and therefore have not been considered sufficiently accurate to determine the level of a particular trace metal consumed by a subject.
The problems with previous hair analysis techniques have caused reliance on urine and blood analysis techniques for the detection of ingested chemicals, e.g~, drugs-of-abuse, medications and toxic chemicals, in a subject. However, 2O these techniques also are lciown to be disadvantageous in that the duration and intensity of use or exposure cannot be ascertained. Urine and blood analysis, at best, can provide short term information concerning ingested drugs or chemicals such as drugs-of-abuse. In addition, there are also problems with the interpretation of such results. For exaatple, the detection of a low level of ingested cheaical in the urine could mean that a subject ingested a small amount of the drug or chemical very recently or a larger amount several days earlier. Thus, chronic drug use cannot be detzrmined wit: these methcds without repeated testing.
In response to the problems of establishing a reliable and accurate method that would measure both the duration and intensity of use of drugs-of-abuse, medications, toxic che:~icals, etc. , ~roric rerferaed by Dr. Werner ~.
3aumgartner, as repor=ed in "Radioinmunoassay of Hair for Dete~ fining Opia;.a Abuse Histories", J. Nucl Med 20:749-i~2 SI~STi~UTE SHEET (RULE 26) ...., .,.,.. ,. . ::,. . ";.. , ,...,..;. ~ . ..
WO 94/18561 ~ ~ PCT/US94/01137 (1979), determined that long-term histories of exposure to drugs-of-abuse can be obtained through the analysis of mamaalian body hair, since these substances are "trapped"
within individual hair fibers during their synthesis. In this respect, hair was shown to act like a tape recorder, i.e., past exposure histories can be evaluated through sectional analysis of hair samples. It was found that heroin, once in the bloodstream, will find its way into hair as it is synthesised.
Thus, it was discovered in this study and confirmed by subsequent studies that a variety of chemicals, such as drugs-of-abuse, medications, toxic chemicals, etc., hereinafter collectively rererred to as "analyte", are trapped by hair during its synthesis and that these substances are "locked up" in hair for essentially the duration of the hair. This was found to be true for head and body hair as well as for other keratinized structures such as fingernails. Suzuki et al., Forensic Sci.
International, 24:9-16, 1984 . These Entrapped substances cannot be washed out of hair, and are completely released only upon the complete, or nearly complete, destruction of the hair fiber.
Prior art methods of extracting an analyte from hair included subjecting the hair to hot methanol solutions (Baumgartner et al., J. Nucl Med 20, 743, 1979) and by overnight incubation of hair in an alkaline or acid medium.
D. Valents, et al., Clinical Chemistry, 1952, Vol. 27, No.
11, 1981. Prior methods also include the use of a mortar and pestle to release the entrapped analyte in conjunction with a solvent.
However, solvent extraction procedures suffer from several problems in accurately determining the presence and amount of an inaested analyte. One of these problems is that the solvent extrac'ion methods frecuent'_y re:~cve only a s:lall S'UBSTmJ~E SHEET (RULE 26) WO 9/18561 . ~ ' PCTlUS94/01137 213350' unk.-gown and variable fr3c~ion of the total analyte present in the hair sample. Such methods also tend to be time consuaing, and generally involve elevated temperatures which may damage the analyte. Another disadvantage is that different analytes require different solvents for extraction. For example, a hair sample containing morphine, phencyclidine ("PCP"), cocaine and marijuana has to be extracted sequentially with several different solvents, which is a very time consuming procedure, particularly since the solvents have to be evaporated before analysis can proceed.
Other methods and studies pertaining to the degradation of hair and hair analysis include:
..
o. Suzuki, et. al., in a publication by Elsevier Scientific Publishers Ireland Ltd., discloses a method for detecting methamphetamine and amphetamine in nail clippings or hair in which the substance was first washed in a~mixture of methanol and water and dissolved in sodium hydroxide, followed by analysis of the extracted drug.
A.W. Holmes, in Textile Research Journal, 706-712, August 1964, discloses the degradation of human hair by papain using sodium sulfite as enzyme activator.
Annette M. Baumcarvr~er. et al., in the Journal of Nuclear Medicine, 20:743-752, 1979, discloses the extraction of morphine and heroin from hair by pulverizing hair with a mortar and pestle followed by treatment with methanol.
D. yalen~te. et e?., in Clinical Chemistry, Vol. 27, No. 11, 1981, discloses Dr. Baumgartner's tec:znique of subjecting hair to a treatrent of hot methanol to effectuate extraction of drugs of abuse as well as the author's tec:~nic_ue o::
extracting mor~:hi:~e in an acid or alkaline medium.
$UBSITTUTE SKEET (RULE 26) WO 94/18561 ~ ~ ~ ~ 5 s o PCT/US94l01137 A.M. Baumaartner, et a~.~, in Journal of Forensic Sciences, p. 576-81, July 1981, discloses the extraction of PCP with mortar and pestle followed by treatment with methanol. The extracted PCP was then analyzed with RIA.
~ 5 smith et al., in Journal of Forensic Sciences, Vol. 26, No.

3, July 1981, pp. 582 -586, disclose the testing of hair for the presence of phenobarbital, in which a single head hair was washed, dried, cut in 2 mm lengths and added to 0.2 ml 0.1% SDS/saline solution, and a sample assayed by radioimmunoassay.
W.A. Bau~ncartner, Black. et al., in J. Nucl Med 23: 790-892, 1982, discloses the extraction of cocaine from hair samples by refluxing the hair samples in ethanol followed by RIA
analysis.
_, in Journal of Forensic Sciences, Vol. 28, No. 2, April 1983, pp. 380-385, disclose a method whereby hair from methamphetamine addicts was dissolved using 1.5 N
hydrochloric acid at a pH between 1 and 2, followed by analysis using a gas chromatograph and mass spectrometry.
&. Puschel. et al., in Forensic Science International, 21 (1983) 181-186, discloses the dissolving of hair samples by exposure to sodium hydroxide and heat followed by analysis for the presence of morphine by RIA.
O. Suzuk~'_~, et al., in Journal of Forensic Sciences, Vol. 29, No. 2, April 198, pp. 611-617, discloses the detec;.ion of methamphetamine and araphetanine in a single hu~aan hai~ by gas c:~romatography and cheaical ionization mass spectrometry. The hai= sa~aple was fi4st dissolved in a sodium hydroxide solution to whic:~ ~aas added N-methylbenzylaaine.
S
SUBSTTI"UTE SHEET (RULE 26) ' . , ~ , . , , . . .. ...r.. . ,.. ... . .. . . . . 'A:'~.'a . . . , . ..,..
. .. . ! ~.: . , . . ..,. ..
WO 94/18561 , , . , PCT/US94/O1137 N.J. Halev et al., in Chin. Che:n. 31/10, 1598-1600 (1985), discloses the analysis of hair for nicotine and cotinine, in which washed hair samples were dissolved in a buffet solution containing gelatin, sodium chloride, Tris and EDTA, and adjusted to pH 7.4. Samples were then analyzed by radioimmunoassay.
Sramek. Baumgartner. et al., in A.M.J. Psychiatry 142:8, August 1985, discloses the analysis of hair samples of psychiatric patients with methanol extraction and radioimmunoassay.
HaumQartner. et al., in Clinical Nuclear Medicine, vol. 10, September 1985, discloses the benefits of extracting entrapped drugs of abuse from hair followed by RIA analysis.
Gill. et al., in Nature, Vol. 318, p. 577 (1585) discloses the use of an SDS/proteinase k/dithiothreital mixture to extract DNA from whole blood, whole semen, vaginal fluid, ZO hair roots, bloodstains and semen stains. The article states that "no DNA could be isolated from hair shafts".
Smith et al., in J. Forensic Sci. 1986, 31(4), 1269-?3, discloses the detection of cocaine in perspiration, menstrual blood stains and hair using RIA.
M-Maraio, et al., in "Deteraination of Morphine and Other Opioids in the Hair of Heroin Addicts by HPLC and MS/MS" at the International Conference, University of Verona, June 3025-26, 1986, discloses various methods to assay morphine from hair samples.
M. Marino. et al., in the Journal of Analytical Toxicology, Vol. Z0, July/August 1986, discloses a methcd for tre quantitative determination of morphine contained in the hair of heroin addicts, by means of heat-acid hydrolysis, O
Sl~$~TrUTE SHEET (RULE 26) WO 94/18561 t 21~ 3~ e7 ~'s ~ PCT/US94t01137 pre-column dansyl derivatization, straight phase liquid chromatography and fluorescence detection.
Smith, et al., in Journal of Forensic Sciences, Vol. 31, No.

4, October 1986, pp. 1269-1273, disclose a method for the analysis of hair for the presence of drugs whereby hair ' samples were first washed, cut into small segments, mechanically pulverized for six minutes,. refluxed in ethanol and the samples analyzed using radioimaunoassay.
M. Michalodinitrakis, Med.Sci.Law (1987), Vol. 27, No. I, discloses the detection of cocaine in rats from the analysis of hair samples, which were dissolved upon exposure to 1.5 N
HCL, which brought the pH value to 1-2, following incubation With 0.01 N HC1 at 37'C for one hour.
Pelf,.. et ai., in Biomedical and Environmental Mass Spectrometry, Vol. 14, 63-63 (1987) discloses a procedure for the identification of morphine in the hair of heroin addicts in which hair is treated with diethylether and hydrochloric acid followed by dissolution of the dried extract in methanol.
HiQllChl-'et al., in Naturs, Vol. 332, p. 543 (1988) disclose a method for dissolving hair at pH 8 by the action of dithiothreitol, proteinase K, and 2% sodium dodecylsulfate in order to extract DNA from the digest by a complex chemical extraction method.
Also noted are certain patents, e.a., U.S. Patent Nos.
3,986,926, 3,960,551, 3,939,040 and 3,623,9x0, which pertain to depilatory agents for the tanning of hides, and disclose , the use of certain enzynes, including papain, in the dehairirg process.
. 35 Howeme~, these and other prior ar~ methods have p~aven disadvantageous~for the reasons noted above and/or because $~Sf tTUTE SHEET (RULE 26) WO 94/18561 21 ~ ~ ~ 6 0 PCT/US94/01137 . ;v .
they degrade the analyte probes (e.Q., antibodies) of biological analytical methods, thereby preventing the use of such highly sensitive analytical techniques.
Thus, there exists a need for an analyte detection method that can rapidly and completely solubilize a certain analyte from keratinized structures of the body such as hair, ' fingernails, toenails and skin of a subject and which permits direct analysis of the identity of the analyte and the duration of use of the analyte in, or exposure to, a subject, wit.'~out destroying the analyte of interest and/or an analyte probe of biological. analytical methods.
SD~iARY OF THE INVENTION
It is an object of the invention to provide a drug and chemical detection method:
It is another object of the invention to provide a drug and chemical hair analysis method;
It is another object of the invention to provide a reliable method of solubilizing and directly analyzing the identity of analytes contained in head and body hair and other 2S keratinized structures of the body, and, where applicable, of determining the duration and extent of exposure of the analyte in a subject;
It is yet another object of the invention to provide a hair analysis method that solubilizes an analyte from the inner core of hair without causing damage to the analyte;
It is yet another object of the invention to provide a reliable hair solubilization and direct analyte detection method that effectively permits the use of highly accurate biological analytical methods suc:~ as radioimmunoassay;

1~V0 94/18561 ~ ~ ~ 3 5 6 0 pCTIUS94/01137 It is yet another object of the invention to provide a reliable hair analysis method that may be perfarmed in a much less period of time than known hair analysis methods.
It is yet another object of the invention to provide a method for removing an interfering cross reacting substance _.. , from a hair sample which may result in false positive results in a marijuana assay.
These and other objects are achieved by the novel analysis method according to the invention, which comprises preparing a mixture containing dithiothreitol (DTT) or .
dithioerythritol (DTE), an enzyme suitable for the dissolution of keratinized structures and a sample of a keratinized structure; permitting DTT or DTE to act=vate the keratinized structure and/or the enzyme; permitting the enzyme to at least substantially dissolve the sample of keratinized stzucture to fona a keratin digest solution: and subjecting a portion of the keratin digest solution to analysis to detect the identity and amount of the analyte, if present, in the keratinized structure sample.
The preferred keratinized structure is hair. The enzyme may be a protease and preferably is papaia, chymopapain, or proteinase K. In order to accelerate the process, copper, e:a., in the fora of cupric sulfate, or sodium arsenite (Na2AsOZ) may be added to the digest solution to deactivate excess dithiothreitol or dithioerythritol in the mixture which may interfere with var=ous components of the iaixture.
Preferably, the analysis of t_~xe solubil ized analyte is performed by a biological analytical method suc:~ as an immunoassay, e.a., radioimmunoassay.

SI~SITIUTE SHEET (RULE 26) WO 94/18561 ' ~ ~ ~ ' ~ PCT/US94/01137 213 ~~:g, 0. , DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, a method is provided that permits the rapid and complete solubilization of a certain analyte from head or body hair or other keratinized structures of an individual who has previously been exposed to the analyte, e~Q., has ingested the analyte,followed by the identification of the analyte by known analytical biological probes, such as the rapid and highly sensitive immunoassays. The solubilization of the analyte from the interior of hair is effectuated without damaging the analyte trapped within the organic matrix of the hair fiber which is to be analyzed, nor does it affect a subsequently-used probe (e. a., antibody) of a biological l5~analytical method. The hair analysis method according to the invention also permits the detection of past use patterns in a subject over extended periods of time without performing repeated testing as is necessary in conventional analyte detection met.~ods which measure the content of the analyte in samples of blood or urine.
More particularly, the invention comprises the rapid enzymatic digestion of the proteins making up samples of hair and other keratinized structures, followed by the effective deactivation of the enzyme and associated enzyme/substrate activator (DTT and DTE). The resultant solubilized analyze in the hair digest solution may then be analyzed by known biological analytical probes, preferably by highly sensitive protein-based analytical technicl:es such as immunoassay. It has been found that the amount or analyze entrapped in hair is directly proportional to the amount of analyze ingested.
In accordance with the invention, a sanple of a keratinized str-ucLure, a.a. , hair, is ffirst callected fron a subject suspected of having been exposed ~o, or having ingested, a par ticular. analyts. Preferably, the hair sa.~"pl.a is f_rst IO
S~STf M'E SHEET (RULE 26}

213 3 ~:6~Q .~.
washed by known methods to remove analyte or other drug or chemical which may have been deposited on the surface of the hair by external contact rather than by actual consumption.
The hair sample is then subjected to treatment with ~ 5 particular enzymes, together with a particular enzyme/substrate activator, so as to effectuate the complete or nearly complete digestion of the organic matrix of the hair fiber, known as keratin. The subject analyte that has been "entrapped" within the organic matrix of the hair is then released into solution, or even if protein bound, the analyte is accessible to the antibody employed in . protein-based analytical methods. In order to fully and accurately carry out the method~aecording to the invention, a complete digestion of the keratinized structure is desirable.
Proteases are preferred for the digestion of the hair samples. Most active, and therefore preferred for use in the invention, are the enzymes papain, chymopapain and proteinase K.
A number of other proteases have been found to be effective in the method according to the invention at low pH values (e-a., pH 7-9), namely, protease Type Iv (bacterial, from Streptomyces caespitosus), Type VIII (from Bacillus subtilis), Type XI (proteinase K, fungal, from Tritirachium album), Type XIV (pronase, from Streptomyces griseus),~Type XVI (from bacillus subtilis), Type XVIII (Newlase, from Rhizopus species), Type XIX (from Aspergillus sojae), Type XYI (frcrs Streptomyces griseus) , Type XXTV (bacterial) , Type X.YVII (Nagarase), Type III (Prolase) and Type XXIII (from Aspergillus Oryzae) (all available from Sigma Chemical Co., St. Louis, MO) .
As noted above, certain art-recognized procedures prcvide for the use of papain for use as a hair depilatory. These depilatory methods remove hair from :tides and skin by 1?
SUBS?ITUTE SHEET (RULE 26) WO 94/18561 ; ~~i~~~ ~.~ p PCTIUS9~10113~
softening it sufficiently so as to per:ait its ready removal by scraping or other mechanical means, and utilize inexpensive and less effective sulfhydryl enzyme and substrate activators such as thioglycolic acid or cysteine.
These methods only partly degrade the hair and do nat provide for the complete chemical digestion of the hair. A
mere softening of the hair does not lead to the conplete, or nearly complete, digestion of hair which is necessar,~ in order to obtain a complete release of "entrapped" analyte.
l0 Moreover, the sulfhydryl enzyme activators used in these depilatory methods are also haruful to certain biological analyte probes such as antibodies.
In contrast to these depilatory methods, the method of the present invention utilizes dithiothreitol ("DTT°', or 2,3-dihydroxybutane-1,4-dithiol) or its isomer dithioerythritol ("DTE", or 2,3 dihydroxybutane-1,4-dithiol) as the substrate and enzyme activating agent. Surprisingly, it has been found that DTT and DT~ produce a highly active enzyme capable of digesting hair within a relatively short period of time, e.a., about three hours, resulting in the release of the analyte into the hair digest solution. This high activity of the enzyme has been found to be due, at least in part, to the activation of the keratinized structure substrate itself by DTT and DTE, presumably by the action of DTT and DTy in opening up disulfide bonds in the keratinized structure, which facilitates enzymatic attack.
Once the protein of the keratinized structure has been completely or at least substantially digested, thereby releasing the analyze into the s;.?ution mixture, it has been found to be necessary to deactivate the enzyme and tZ~
enzyme/substrate activators) in crder to subject tre analyze to biological analytical probes such as an4'_odies, since the enz "e and enzyme/subs~~ate activatcr(s), as noted above, can interfere xith the st=t~ctural integrity c;
protein substances involved in t::e analytical :aethec.

51~5'~'1UT'E SHEET (RULE 26) WO 94/18561 . 0 PCT/US94/01137 The task of deactivating the sulfhydryl-dependent enzymes such as papain has proven difficult since after the hair digestion step, the enzymes are "buried" in a "sea" of sulfhydryl groups belonging to the released hair proteins and enzyme/substrate activating agents. Known sulfhydryl blocking agents are ineffective in deactivating the enzymes, since the known sulfhydryl blockers tend to bind to the degraded hair proteins and DTT or DTE and not necessarily to the enzyme sulfhydryl sites critical for blocking the activity of the enzymes. Thus, it is not possible to effectively utilize the protein-based analytical methods if the enzyme_sulfydryl sites are still active.
Thus, it was surprisingly discavered that DTT and DTE act not only to activate enzymes and/or the keratinized structure substrate causing unexpectedly high hair digestion activity, but that they also may act to deactivate the enzyme by a direct or indirect (enzyme self-deactivation) mechanism after the enzyme effectuates the complete, or nearly complete, digestion of the hair protein. Typically, the enzyme deactivation occurs within about four to five hours after exposure of the DTT or DTE to the enzyme, which is a sufficient amount of time for the enzyme to effectuate the digestion of the hair sample. Once the enzyme has been deactivated, it has been found that the enzyme cannot be reactivated or regenerated by exposure to fresh DTT or DTE.
Deactivation of at least certain of the non-sulfhydryl dependent proteases, e.a., proteinase K, by its inhibitor, phenylmezhyl sulfony? chloride, is generally not re~uirad since the enzyme has not been found to be active against the antibodies used in protein based immunoassay tech~icues.
It has also been found that ac~ive DTT and DTE prese:~t in the hair digest soluticn const?tute a hazard to the st=-ucture and ac~ivit~J ez other p~eteizs to :~hich _.. is exposed, e.c., _ntibodies utilized in radioi-,,unoassay.

SUIBS1TTUTE SHEET (RULE 26) WO 94118561 . . . PCTIUS94l01137 Thus, it was a further surprising result that DTT or DTE in the reaction mixture may not only acs to deactivate the enzyme, but itself becomes deactivated in the digest solution without the introduction of an inhibitor.
Typically, the spontaneous deactivation of DTT or DTE will occur after the hair sample has been digested but less than about 14 hours after its first exposure to the enzyme, depending on the various concentrations and amounts of the enzyme and DTT or DTE utilized, the pH, temperature, amount of hair sample, etc.
Thus, in accordance with the method of the invention, complete hair digestion can be carried out in a relatively short period of time, e.~, overnight, and the hair digest solution, which includes the released analyte of interest, can be directly subjected, effectively and accurately, to protein-based ligand assay analysis methods the next morning. Typically, the entire method, from the washing of hair samples to the identification of the analyte, should take no longer than about 16-20 hours. Little or no intervention by the individual performing~the method is needed to release the analyte from the hair sample once the enzyme and DTT or DTE come into contact with the hair sample.
Alternatively, it has been discovered that the addition of copper, preferably in the form of cupric sulfate or other similar compound which produces copper ions in solution, to the sulfhydryl grcup-ric'~ hai:° digest solution acts to more rapidly deactivate the sulfhydrrl groups of DTT or DTE.
Thus, the addition of low amounts of the cubric sulfate to the hair~digest mixture after digestion of the hair sample and the deact~.vation of the enz ..e by DTT or DTE
significantly accelerates the tire in which the hair digest mixture can be subjected to the ir.iaunoassay method since it is not necessary to wai~ for the ceactwation of CST or DT=' -. , which typical?y occurs ?ess than a:,out Fourteen zcu~s ofra=
,s S'UBSiTTUI'E SHEET (RULE 26) WO 94118561 ,, PCT/US94101137 its addition to the digest solution. Typically, about tuu microliters of cupric sulfate (10 mg/mi) is added to 1 ml of hair digest solution about 4 to 5 hours after contacting the enzyme and (DTT or DTE) with the hair sample so as to permit the enzyme sufficient time to dissolve the hair sample.
Similarly, sodium arsenate (NaAs02) may be utilized in the invention to remove residual DTT or DTE by formation of a precipitable compound. Typically, I00 microliters of a 100 mg/ml solution of sodium arsenate is added to I ml of hair digest solution to effectuate the deactivation of DTT and DTE.
Once the rapid and effective dissolution of hair for the purpose of releasing entrapped analyte is effectuated as -described above, the analyte mixture may then be subjected to direct analysis by art recognized protein-based analytical methods such as radioimmunoassay (°°RIA"). Such methods are preferred for use in the invention because RIA
and related immuno- or ligand assays are currently the only known mass production procedures having the required sensitivity and convenience for measuring the low concentrations of analytes contained in hair samples. The use of these methods is preferred because only about 0.5 to 1.0 mg. of hair is necessary for analysis by RIA and other protein-based analytical methods. Indeed, for certain drugs-of-abuse, it has been found that analysis by the method according to the invention can be effectively ~0 perforaed on as little as one or tyro hairs about one inch in length.
Other analytical methods may be uti~.ized in place of the protein-based analytical methods, including instru.:~ental 3 5 means suca as c::r omatography, mass spec ty cmetr l, a c .
Because these met:~ods are not grotei:z-based, the seeps of deactivation cf the enz_~~e and DT': or DTE is pct necessary '? 5 'UtE SHEET (RULE 26) when using non-protein-based analytical techniques.
However, the speed and gentleness of the extraction method according to the invention and the ability to quantitate the extraction efficiency through the inclusion of a "spike", iie., the inclusion of a known amount of analyte, makes the presently disclosed digestion method also the method of choice for instrumental analysis methods such as gas c.'lromatography and mass spectrometry.
The method according to the invention has been found to be effective in detecting the use and prior use of drugs of abuse such as cocaine, morphine/heroin, marijuana, .
phencyclidine or "PCP", methaqualone and methamphetamine.
Moreover, the method according to the invention has been found to be effective in determining prior usage of prescription drugs such as digoxin and amphetamines and toxic chemicals such as nicotine. It is contemplated that any organic analyte present in the bloodstream of an individual which is transferred to the hair during its, synthesis can be extracted and analyzed in accordance with the met.'~od of the invention.
In carrying out the method, it is preferred that an acrueous solution of about 110 mg DTT or DTE/10 ml water be used, although concentrations of DTT or DTE of about 50-200 mg/10 ml water are effective in the method. It is preferred that the weight ratio of DTT or DTE to papain or chymopapain be about 110:2 [when enzyme purity is 16-40 EAEE units/mg protein], although efficacious results have been observed at weight ratios of OTT or DTE to papain or chymopapain ranging bet~reen about 110:1 to about 110:4. With respect to protei:nase K anc other proteases, it is preferred that the weight ratio of DTT or DT~ to proteinase K (or ot::er proteases) be about 1200:1 (when enzyme purity is ~0-~0 units per tag. protein), although ;eight ratios of .200:0.5 to about 1200:2 -.gill also be effective.
16 .
SUHS~(1'UTE SHEET (RULE 26) WO 94II8561 21 ~ 3 ~ ~ ~ PCT/US94/01137 The concentration of hair protein is preferably kept constant at about 10 mg hair /cc of digest solution so as to prevent variable matrix effects in a subsequently utilized protein-based analytical method.
It is preferred that the enzymatic digestion of hair, according to the method of the invention, be conducted at low temperatures and near neutral pH. In this regard, it is preferred to perform the method when pepsin, chymopapain or other sulfhydryl dependent enzyme is utilized as the enzyme, at a temperature of between about 20'C and 40'C, and at a pH
between about pH 8.8 and 10.5. Preferably, the pH of the method is between about 8.8 and 9.5. In a most preferred embodiment, the temperature is about 37'C and the pH about 1 S 3~.1.
When proteinase K or other proteases are utilized as the enzyme, it is preferred to perform the method between about and 40 degrees centigrade and at a pH between about 7 and 20 9. In the most preferred embodiment, the temperature is about 37 degrees centigrade and the pH about 7.0: under these conditions, the risk of damaging a particular analyte is at a ainimum. Other enzymes which dissolve hair under neutral or acid conditions include: Protease Type XIV
(Fronase), Type III (Prolase), Type IV, Tvpe VIII, Type XVI, Type XVIII, Type XIX, Type XXIV, Type XXVII (Nagarse), Type XXVIII, Type XXI and Type XXIII.
Under certain circumstances, it is advantageous to perform the method according to the invention at a lower ~'~an usual pH in order to preserve the chemical structure of the analyte or to simplify the per=ormance of the method.' As stated above, the digestion of hair will occur at a pH
bet~reen about 8.3 and 10.5 when a sulfhyd.:yl dependent . ' 35 enzyme (e.c., pepsin) is utilized and between 7 and 9 when a protease (ela. , Proteinase K) is utilized. r1t any ~aFi in either of these ranges, however , certain analytes mar beco:,te SI~STOI~TE SHEET (RULE 26) unstable or hydrolyze to a different fore, which may lmpacE
on the measure of both quantity and quality of the analyte in the subsequent analysis step.
For example, cocaine will hydrolyze to benzoylecgonine at a pH above about 6.6 and a temperature of 37'C. Thus, infested cocaine naturally will hydrolyze to benzoylecgonine in the blood and eventually end up entrapped in hair as benzoylecogonine, and the digestion of hair from a cocaine user will lead to the release of the benzoylecgonine from the hair into solution thereby permitting a positive identification of cocaine ingestion by immunoassay. The method of the invention thus actually measures the amount oz benzoylecgonine, rather than the amount of cocaine, that has been incorporated into the hair from blood. However, a groblem may arise if there is inadequate washing of the hair sample prior to digestion if the digestion is performed at a pH greater than about 6.6 in that a false positive result may be obtained if the individual being tested was exposed to atmospheric cocaine that has settled on the surface of the inadequately washed hair sample.
Thus, in the case of certain analytes such as cocaine which may be chemically altered by a higher pH, it is desirable to perform the method of the invention at a pH which avoids hydrolysis or other chemical reaction of atmospheric analyte which inadvertently has ended up in the digest~solution. In the case of cocaine, performance of the method at a pH below about 6.6 at 37'C will ensure that the benzoylecgonine in the sample is directly related to the amount of cocaine ingested by the subject.
according to the invention it has been found that cer~ain biological detergent compounds useful for solubilizing biolcgical me::brane components aid in the digestion or hair at a relativel y ' ow pH while nom interfering :,rit:~ enzymatic acti~rity or the antibody-antigen reaction ~rhic:~ will to S~'1TTUTE SHEET (RULE 26~

WO 94118561 ~ , PCT/US94It11137 influence the sensitivity of the immunoassay. This is surprising and unexpected since other biological detergents have been found to be unsuitable for use in the invention because they are ineffective in aiding digestion at the desired low pH, they deactivate proteinase K or other hair protein digestion enzymes and/or they impact on the binding of the analyte by the antibody thereby drastically reducing the sensitivity of the immunoassay. See, e.a., Hicuchi, R.
et al., "DNA Typing From Single Hairs", Nature, 332:543-546, 1988 .
The biological detergents together with an appropriate enzyme (e. a., protease and sulfhydryl enzymes) and activator (era., DTT and DTE), are effective in aiding the digestion of the hair sample at a lowered pH in the range of about 6.2 and 8. Certain of the biological detergents described herein are effective in the invention at a pH as low as about 5.8.
The protease enzymes (e-c :,, proteinase K) are preferred for use with the biological detergents disclosed herein. The biological detergents also effectively may be used together with the sulfhydryl enzymes, e.c., papain, to effectuate digestion of a hair sample at a lower pH.
The biological detergents found to be useful in the invention include the bile acid detergents, such as glycocholic acid, cholic acid, taurocholic acid, deoxycholic acid, glycodeoxyc:~olic acid, taurodeoxycholic acid and salts thereof, including sodium salts. Other detergents effective for use in the invention are sul~o-betaines, such as the Zwittergentsa, and betaines, suc:~ as Enpigen BB (N-dodecyl-N,N dir,ethylglycine) (all ava=lable from Calbioche:~ Corp. , La Jolla, Ca).
3~
Still other detergents whic:~ are useful in aiding the digestion of hai. according to t::e invention at a rgla~iveiy S~1'OUT'E SHEET (RULE 26) lower pH are the alkylglucosides, including hexyl-5-D-glucopyranoside, heptyl-B-D-glucopyranoside, octyl-B-D-glucopyranoside, nonyl-B-D-glucopyranoside, decyl-B-D-glucopyranoside, dodecyl-B-D-maltoside and octyl-H-D-thioglucopyranoside (OSGP). Mixtures of alkylglucasides, such as the product ELUGENT (Calbiochem), are also effective.
Particularly preferred for use in the invention are the bile aeids cholic acid and glycocholic acid which aid in the digestion of hair at a pH in the range of about 6.3-8. The deoxycholates such as deoxycholic acid and glycodeaxycholic acid are effective in aiding in the digestion of hair at a pH above about 7.
~~
As discussed above, the hydrolysis of cocaine occurs at a pH
of about 6.6 and above at 37'C. To avoid hydrolysis of cocaine to benzoylecgonine, the hair digestion preferably is performed at a pH in the range of about 6.2 - 6.5. To this end, the cholate detergents are preferred over the deoxycholate detergents which effectuate the digestion at a somewhat higher pH.
Of the sulfo-betaine detergents manufactured by Calbiochem Corp. of La Jolla, CA., Zwittergent°' SH3-14 (CAS Registry No. 14933-09-6, N-tetradecylsulfobetaine or 3-(dodecyldimethylammonia) propane-1-sulfonate) is preferred. Digestion of hair using the Zwittergent~
sulfo-betaine detergents typically occurs at about pH 6.3 at 37'C. Zwittergents' are of the class of detergents known as sulfo-betaines having the general strc:cture:
9N~Sf ITUT'E SHEET (RULE 26) :. _.._ _ _ _~._.. .,~ ,, . .:. , ~. -.. .. ,: . . ,_ . . .... . ....... .. .
. _. ..
. . . : ~- ..,~.... . ._z... . ~ . . . . .. ..~~ ~. . . : .. . . ,. _..

WO 94li8551 ., P~CT/US94/01137 Ca3 .

(+) II
CH3(CH2)x-N-(CH2)3-S-0(-) ~3 wherein x may be any number which provides an effective biological detergent. Preferred are.those compounds wherein x is in the range of 7-16. Most preferred is the detergent when x is 14. Other Zwittergent~ sulfo-betaine detergents useful in the invention include:
1. Zwittergent' SB3-08 wherein x=7 [N-octyl sulfo betaine gr 3-(Octyldimethylammonio) propane -1-sulfonate] [CAS
Registry n 15178-76-4].
2. Zwittergent~ SB3-10 wherein x=9 [N-D-dodecylsulfobetaine or 3-(dodecyldimethylammonio) propane - 1 - sulfonate] [CAS
Registry ~ 15163-36-7].
3. Zwittergent~ SB3-12 wherein x=11 [N-dodecylsulfobetaine or 3-(Dodecyldimethylammonio) propane - 1 - sulfonate] [CAS
~14~33-~9-s].
4. Zwittergent' SB3-14 wherein x=13 [N=tetradecylsulfobetaine or 3-(Dodecyldimethylammonio) propane - 1 - sulfonate] [CAS T 14933-09-6J.

5. Zwittergent~ SB3-16 wherein x='_5 [N-Hexadecylsulfobetaine or 3-(Hexadecyldinethylam..:~onio) 3~ propane -- 1 - sulfonate] [CAS 12281-11-0]
- The bile acid detergents and Caibioche~ Z~sittergents~ SB3-3, S3-10, S33-12, SB3-14 and S83-16 (x in t:~e range oL 7-16) ' are preferred in effectuating the digest=on of hai= at a rel atively lower pH whica is desirable in zet::a:t:pi:ets:aine, 2, suesTnttt~ sHE~r ~u~E ~s~

WO 94/18561 . , PCT/US94/01137 ,, PCP and opiates assays. The cholate and c~albiochem Zwittergents~ are preferred for use in the cocaine assay.
The cholate and deoxyeholate detergents are preferred for use in marijuana screening assays.
In practice, the biological detergent is mixed with the aqueous digest solution of the activator such as DTT (or DTE) and the enzyme (preferably proteinase K) prior to contact of the solution with the hair sample at a preferred temperature range of about 30-~0'C as described herein.
Typically, about 1-2 mg of biological detergent is added to about 1 cc of digest. solution.
In another embodiment according to the invention, an ion 15-exchange resin is employed to remove from the hair digest solution a substance which uniquely interferes with the marijuana assay. This interference has occurred with all currently available commercial RIA kits suitable for detecting cannabinoids. The interfering substance, effectively present in every hair sample in varying amounts from individual to individual and believed to be naturally occurring in hair, appears to interfere with the assay as a result of a cross reaction with the marijuana RIa antibody (3,.e_., specific to cannabinoids) rather than by matrix effects. This appears to be the case because dilution of the interfering substance produces an asymptotic curve which appears identical in shape to the calibration curve obtained with the earboxytetrahydro-cannabinol ("carboxy-TFiC") standard used for an RIa marijuana assay, rather than producing an S-s:.aped dilution curse which would have been expected if matrix effec~s were the cause of the interference. It appears that this interfering substance is lipidic and bears a close resemblance in structure to cap 5oxy-TFiC .
Because of its ~iailarity to car~ox=r-THC, and ether cannabinoids suca as tet~aryd~~caznabinol ("THC"), the $UB:STTf UTE SHEET (RULE 26) 2133 5~ fi Q
interfering substance results in false positive results in assays on hair digests using RIA to determine marijuana exposure. In other words, the RIA erroneously will identify the interfering substance as a cannabinoid from exposure to marijuana, even in individuals not exposed to marijuana.
Thus, it is necessary when performing an RIA assay for - marijuana exposure to somehow remove the interfering substance from the digest solution prior to subjecting the solution to immunoassay analysis.
Since the presence of this interfering substance in hair is a new discovery, there is no method known to the art for removing it. Removal of the interfering substance from the digest solution is further complicated by its similarity to 15"lipidic carboxy-THC and THC, the more common diagnostic analytes in a marijuana assay. Many filtering techniques with the capability of filtering out undesirable substances are not effective in removing the interfering substance from the digest solution, because they either do not effectively remove the interfering substance, and/or because they remove the analytes, e_.g., THC and Carboxy-THC.
It was thus surprising that there exists in hair an analyte indicative of marijuana exposure which does not possess many of the same lipidic properties of the interfering cross reacting substance but yet will react with the cannabinoid antibody used in the RIA assay for marijuana. The exact chemical structure of this immunoreactive substance(s), or modified-lipidic marijuana analyte, is unknown. however, its presence in the hair sample will confirm marijuana exposure.
Also surprising is the discovery that the interfe~~ng substance may be removed frog t:~e digest salut.on without removing the newly discovered modified-iipidic :gar=juana analyze by t:~e use of cer rain commercially avail a;cl a icn exc:~ange res ins . It has been f ound t:~at suspers icrs o 2' ..
S'UBSTITLITE SHEET (RULE 26) certain ion exchange resins upon contact with the digest solution will remove the interfering substance along with certain cannabinoids such as THC from the digest solution, but will leave in the digest solution other diagnostic cannabinoids, such as the modified lipidic marijuana analyte, which then may be detected by commercially available RIA kits utilizing a cannabinoid antibody. The use of these ions exchange resins to remove the interfering substance, thus permitting the detection of the modified-lipidic marijuana analytes in the RIA
assay without interference, is both convenient and cost effective.
Effective ion exchange resins are both anionic and cationic. They generally are commercially available, such as from Sigma Chemical Co. of St. Louis, Missouri. However, they have been found to be most effective not in the form in which they are commercially available (generally course, fast settling particles), or in the way they are generally used (e.g., packed columns) but when contacted with the digest solution in the form of a suspension, e.g., when broken apart into much smaller, slowly settling particles by vigorous stirring and made into a fine suspension of these small particles. Effective ion exchange resins include:
1. Anion exchangers on dextrose such as DEAE Sephadex (A-25 and A-50 Diethylaminoethyl Sephadex) and QAE Sephadex (TM) (Q-50 Diethyl-[2-hydroxypropyl] aminoethyl Sephadex) (TM);
2. Anion exchangers on agarose such as DEAE Sepharose (TM) CL-6B
(Diethylaminoethyl Sepharose) (TM) and Q Sepharose (TM);
3. Amion exchangers on cellulose such as DEAE-Sephacel (Diethylaminoethyl Sephacel); Ecteola Cellulose (Epichlorohydrin Triethanolamine Cellulose); PEI
Cellulose (Polyethyleneimine Cellulose); QAE Cellulose (Diethyl-[2-Hydroxypropyl] aminoethyl Cellulose); and DEAE cellulose;
4. Cation exchangers on dextran, such as SP Sephadex (TM) C25 (Sulphopropyl Sephadex) (TM);
5. Strongly acidic cation exchangers on polystyrene, such as Amberlite (TM) (active group: sulfonic acid, sodium form); Dowex (TM) HCR-S (active group:
nuclear sulfonic acid, hydrogen form) and Dowex (TM) macroporous resin (active group: nuclear sulfonic acid, hydrogen form);

6. Specialty Exchangers such as benzyl DEAF cellulose (Benzyl Diethylaminoethyl cellulose) and TEAE cellulose (triethylaminocellulose).
The concentration of the resin will vary depending on the resin employed.
Concentrations may vary up to about 50% (wt./vol.). For example, a suspension of DEAE Sephadex (TM) A25 in deionized water preferably is prepared using about 3 to 9 gm. resin-100 ml water. The suspension is allowed to swell. At least one hour of swelling at room temperature has been found to be adequate. After swelling, the suspension is stirred or shaken vigorously for at least thirty minutes and preferably up to about sixty minutes until a very fine suspension is obtained.
Preferably, the suspension has a settling time in the range of 50-60 minutes compared to about 10-15 minutes of an aqueous suspension of an unshaken resin. Vigorous stirring or any other method which results in the break up of the resin into small particles provides the optimum results in removing the interfering substance from the digest solution.
To achieve removal of the interfering substance, approximately equal parts of hair digest solution and fine suspension are mixed together. The ratio of suspension to hair digest solution, however, will vary depending on the resin used and its concentration in the resin suspension.
Generally, the ratio of digest solution to suspension is 4 to 3, if a suspension of 3 to 9°s (wt./vol.) is used. The mixture is rotated so that the suspension and digest solution are in direct contact. Distilled water is then added, the mixture centrifuged and a sample of the supernatant from the resinated digest solution assayed according to the invention.
While the method according to the invention for removing the interfering substance has been described in connection with a digest solution, it is recognized that the ion exchange resin suspension may be used in any hair analysis method where removal of the interfering substance is desirable or necessary, provided that the analvtes are ultimately contained in an aqueous albumin solution near neutral pH as rec;uired for the RIA assay. The resin suspensions may be used, for example, together with known hair extraction (acid, base or solvent) methods or any other method which disrupts a hair sample and results in the release into a solution at least a portion of the contents of a hair sample which includes the interfering substance.
In contrast to other available analyte detection methods such as urine and blood analysis, the method in accordance with the invention permits detection of exposure to an analyte over a period of time, a.rd is therefore quite beneficial in detecting chronic c=-ug use. Since hair is known to grow at a rate of about 0.3-0.4 mm/day o:- about 1.0-1.3 cm/mont!:, it is possible to measure consu:,tation or exposure as far back as the hair length pewits by evaluating snippets of hair of va=ious lengths, and the use of highly sensitive protein-bases analytical methods pe=-nits analysis of s:aal 1 samples of anao_~tp contained in ~::e sw all snippets of hair.
ao St~S'ITTUTE SHEET (RULE 26) WO 94118561 rCT/US94101137 2~3~5so Through sectional analysis, the method of the invention provides a relatively permanent record and evidence of a pattern of drug use, or the prior ingestion of other substances, for periods ranging from several days to months ' 5 or even years after last use. The history of such exposure can be made as detailed as desired by analyzing suitably short sections of hair representing different periods of growth. In this way, prior usage over time, and the extent of such use, can be determined.
Although the use of head hair is preferred for use in the invention due to its length and accessibility, it is possible to utilize any other body hair in the method of the invention. Thus, it is not practically possible to avoid testing by the methcd of the invention by shaving one's head.
However, treatments such as perming and dyeing may increase the rate of dissolution of hair subjected to the method according to the invention. In some cases, some analyte may be lost prior to performing the procedure due to such treatments. When the subject hair has been so altered, an increase in digestion rate is evident and an appropriate correction factor may be applied based upon known rates of normal hair dissolution.
Certain other cosmetic agents, such as certain relaxing agents, may cause hair to become resistant to digestion.
Such resistance may be overcome by increasing the quantity of enzyme to be used. Preferably, proteinase K is utilized as the enzyme Tahen such z~esistance to digestion is encountered.
Alternatively, when it is not possible to make use of body hair or in some instance ~~rhen the use oz hair is nct desi=able, the use of other ke=a~'_nized tissue such as fingernails and toenails may be used in the inventio.~.~.. In S1L~BS"ITTLITE SHEET (RULE 26) this regard, the effective ratio of DTT or DT~ to enzyme needed to dissolve fingernails and toenails in order to release the analyte is about the same as for use with hair, as discsssed above. Once the fingernail or toenail samples are dissolved in accordance with the method described herein, the released analyte may be analyzed by a desired analytical method.
In another aspect of the invention, it has been surprisingly discovered that melanin granules contained in hair can be dissolved by the combined action of the enzyme (preferably papain), DTT or DTE and ethylene diamine tetraacetic acid (EDTA), the latter at a concentration of about 5 mg EDTA/ml of digest solution. Since certain analytes or drugs of abuse such as PC? have been discov eyed to accumulate in these granules, dissolution of the granules, which are present in the digest solution of hair, can be effectuated and the analyte contained in the granule identified.
In accordance with this' aspect of the invention, a hair digest solution is obtained as described above, and the melanin granules recovered from the hair digest solution, e:a-, by centrifugation. The melanin granules are then contacted with EDTA, the enzyme and DTT or DTE to release the analyte from the melanin granules, and the analyte analyzed by the methods described above.
The benefits to be obtained from use of the method accarding to the invention are many. The method provides a prompt and accurate diagnosis of prior exposure to a particular analyte. The subject hair and keratinized structure analysis method can provide a recor3 of consumption, or non-consumpticn, over very long periods of time. Guess work regarding the tr~,:e significance of one blood or urine analysis will be eliminated. Hair collection is less intrusive and less physically repulsive ~han blocd cr urine collec~ion, and samples cannot be altered or subst:.tuted, SI~B~S'tTTUTE SHEET (RULE 26) WO 94118561 ~ 213 3 5 5 0 ~C~~S94/01137 nor can detection be evaded by short tez-~z abstention or "flushing" (excessive fluid intake) prior to a scheduled testing, e.a., pre-employment test or annual physical examination. Samples may be stored indefinitely without refrigeration.
The methods according to the invention, useful for the digestion of keratinized structures, era., hair, can also be used to ascertain the presence and structure of naturally occurring components of hair such as DNA.
The following examples illustrate certain aspects of the invention but they do not limit the invention as set forth in the specification and claims.
15' Extraction of Cocaine From Hair Sample 10 mg of hair was removed from a subject suspected of being a cocaine addict and washed by shaking in water at 37~C for minutes. To IO ml, of distilled water, i10 mg. of dithiothreitol (2,3-dihydroxybutane-1,4-dithiol, Cleland's reagent, obtained from Sigma Chemical Co., St. Louis, MO.), 25 was added. The pH of the solution was adjusted to pH 9.1 with 15% potassium hydroxide added dropwise with stirring of the DTT solution. Stir:-ing was continued while adding 80 microliters of Type III papain solution (papainase EC
3.4.22.2) (ebtained from Sigaa Chemical Co., 16-40 3AEE
30 units activity pe= mg. protein). The enzyme solution was at a concentration of 30 mg of enzyme protein/ml of water, where 1 mg of enzyme protein has an activity of 16-40 BAEE
units [one BAEE unit ~~ill hydrolyze 1.0 taicromole of sodiun benzoyl-L-arginine ethylester at pH o.2 at 25'C].
To 1 ml of this solution Haas added the 10 ng hair sa:~ole in a l3x~S mm polycarbonate test tube. The solution was SUBSTITUTE SHEET (RULE 26) WO 94/18561 " ; : ~ PCT/US94/01137 incubated in a 37'C water bath with shaking for 2 hours, and the solution was allowed to stand overnight at 37'C without shaking. The solution containing the dissolved hair sample was cent::ifuged at 2,000 rpm [Daman IEC model CRU 5,000 centrifuge] to remove the melanin granules. To 1 cc of the hair digest solution Was added 200 microiiters of a 1 molar phosphate buffer, pH 5.5.
100 microliters of this solutian was assayed by RIA for the presence of cocaine [benzoylecgonine equivalent, or "BEE"].
RIA analysis revealed 83.6 nanograms HEE/10 mg of hair.

1'S Addi Lion of Dithioerythritol The hair sample of Example 1 was analyzed using the digestion and assay procedure set forth in Example 1, except for the replacement of dithiothreitol (DTT) by dithioerythritol (DTE). The sample was assayed by RIA, which revealed 82 nanograms cocaine (BEE) per 10 mg of hair.

Addition of Cupric Sulfate After digesting the hair sample in the water bath for four hours, 100 microliters of a l0 mg/ml cupric sulfate solution was added to 1 ml of the hai; digest solution prepared as set forth in Example 1. The solution was shaken at 37'C for about 30 minutes prior to the addition of phosphate buffer and assay by RIA. One hundred microliters of the hair digest solution :,ras subjected to RIB analysis, which revealed 35.0 nanograns of cocaine (BEr)/10 mg of hair.

SU~T!'tUTE SHEET (RULE 26) E'C.~'~!PLE 4 Addition of Sodium Arsenite After digesting the hair sample in the water bath for four hours, 100 microliters of a 100 mg/ml sodium arsenite solution was added to 1.0 ml of the hair digest solution prepared as set forth in Example 1. The solution was shaker.
at 37'C for 30 minutes. 200 microliters of 1M, pH 6.5, phosphate buffer was added prior to assay by RIA. One hundred microliters of the hair digest solution was subjected to RIA analysis, which revealed 32 nanograms of cocaine (BE~) per 10 mg of hair.
15'~ EXAl'4PL~ 5 Substrate Activation By Dithiothreitol (DTT) 10 mg of hair were exposed to 11 mg of DTT at pH 9.1 for a period of 20 hours. The DTT solution was removed and replaced with DTT and papain as in EXAMPLE 1. The hair specimen dissolved within ZO minutes as compared to within one hour for a control specimen not pretreated with DTT and digested as in EXAMPLE 1, thereby demonstrating that DTT
activated not only the sulfhydryl-dependent enzyme, papain, but the enzyme substrate, hair, as well.
F:CAIK~LE 5 Digestion and Analysis of Hair Using Protsinase-K
10 mg of hair was removed from a subject suspected of being a cocaine user and washed by shaking in water at 37 degrees centigrade for 60 ninutes. To 10 ml of 0.05 tZ TRIS buffer, pH 7.0, 60 ng of dithiathreitol (DTT) and 20 mg sodiun dodecyl sulfate (laur~ll sulfa~e) was added. The pig of the solution ~aas c:~ecked to ensure, t:~e solution aas buf=eyed at '1 CUTE SHEET (RULE 26~

a pH 7Ø To this solution was added 0.5 mg proteinase h [Protease Type XI, from Tritirachium album, obtained from Sigma Chemical Co.; 1 mg of enzyme protein has an activity of 10-20 units; one unit will hydrolyze casein to produce color equivalent to 1.0 umole (181 fag) of tyrosine per minute at pH ?.5 at 37 degrees centigrade (color by Folin-Ciocalteu reagent)].
To 1 ml of this solution was added the 10 mg hair sample in a 13 x 75 mm polycarbonate test tube. The solution was incubated in a 37'C water bath with shaking for 1 hour and allowed to stand overnight at 37'C without shaking. The solution containing the dissolved hair sample was centrifuged at 2,000 RP~I to remove the melanin granules. To I5~1 cc of the hair digest solution was added 100 microliters of cupric sulfate penahydrate (10 mg/ml), and this solution was shaken at 37'C for 30 minutes. 200 ul of 1 M phosphate buffer, pH 7.0, was then added.
100 microliters of this solution was assayed by RIa for the presence of cocaine (benzoylecgonine equivalents, or BEE).
RIA analysis revealed 31.2 nanograms BEE/10 mg of hair.
EX~M~LE 7 Role of Sulfhydryl Compounds in the Activation of Hair for Proteinase i~ Digestion 10 mg of hair was incubated in a solution identical to that described in Example 6 except that DTT was omitted. No digestion of hair occurred during 2~ hours of enzy:ae exposure, thereby demonstrating the need for the activation of the hair sample by the subst:-ate activator, DTT.
~2 ~stnuTF sH~~ ~uu ~s) WO 94/18561 PCTIUS94l01137 s 21335sU

Dissolution of Fingernails A 10 mg. sample of fingernail clippings was obtained from a subject, and subjected to a detergent wash. 220 mg of DTT
was added to 10 ml of water in a test tube and the pH
adjusted to pH 9.i as in Example 1. A papain suspension, 160 microliters, was then added. 1.0 ml of this~solution was then added in a test tube to 10 mg of fingernail clippings and shaken at 37~C for a period of 24 hours until dissolution occurred. The digest solution was then analyzed by RIA as previously described.
15' EXrIMPLE 9 Performance of Sectional Analysis A hair sample, about 6 cm in length, was obtained from an individual suspected of being a heroin addict. The samples were carefully sectioned into three 2 cm sections, with corresponding sections added to three separate test tubes and washed. The hair samples were subjected to the process described in Example l, except that chymopapain (EC
3.4.22.6) was used in place of papain as the enzyme. The samples Were agitated overnight as previously described.
RIA analysis revealed morphine content in the three sections of 13.5, 5.7 and 0 nanograms/10 mg hair.
EYA:~!PT_E IO
Dissolution of Digestion-Resistant Hair Ten milligrais of hair Tahich had been treated with ee? axes was incubated overnight in the solution digest descri:.ed in Example 6. The hair sample did not dissolve in t:~e usual S~11T!!TE SHEET (RULE 26) WO 94/18561 . PCT/US94I01137 20-hour period. A greater and additional amount of proteinase K, i.e., 1 mg, was then added to the partially digested sample. The sample then dissolved within the next 24 hours. The digest was centrifuged and 100 ~1 CuS04 solution (10 mg/ml) was added to 1 ml of the supernatant which was then shaken at 37 degrees centigrade for 30 minutes. 200 ul of 1 M phosphate buffer pH 7 was added.
Due to the high amount of proteinase K in the resulting digest, 20 ~1 of the inhibitor phenylmethyl sulfonyl chloride in ethanol was added to the digest prior to assay by RIA.
RIA analysis revealed 7.4 ng cocaine (BE)/lOmg hair.
1~ Example I1 Digestion And Analysis of Hair Using Proteinase K and Cholic Acid Detergent Separate samples of 10 mg of nonaal hair and 10 mg of hair containing the cocaine metabolite benzoylecgonine (BEE) were collected and placed in separate 12 x 75 mm polycarbonate tubes. One ml of the following solution was added to the hair in each of the tubes: 0.5ri Tris buffer (pH 6.5 at room temperature) containing one ml 2 U Proteinase K, 20 mg cholic acid (sodium salt) and 60 mg dithiothreitol. The mixtures were shaken 16 hours at 37'C. After 16 hours, the samples were centrifuged at 3,000 RPM for 20 minutes. To 0.9 ml of the suaernatant removed after centriugation was 3o added 10 ~xl of phenylmethylsulfonyl fluoride (6~ in Ethanol). This was mixed, followed by the addition of 90 ul CuS04. 5ai20 (10 g:;:/liter) to each solution. This mixture was shaken at 37'C for 30 minutes. 100 ~1 of this mixture was then assayed by RIa. Ecuivalent solutions as described but containing no cholic ae_d detergent and no hair protein were also assayed. The results ~~rere as follows:
SU~~ SHEET (RULE 26) WO 94118561 J ~ PCTli1S94/01137 A. Sample of hair protsin not containing cholic acid or BEE: 14,686 cpm (counts per minutes on gamma counter) (Bo) .
Sample of hair protein containing 0.14 ng BEE and no cholic acid: 11,128 cpm (B), or a B/Bo of ?6% (indicating the presence of BEE).
B. Sample of hair containing cholic acid but no BEE:
12,048 cpm (Bo) Sample of hair containing 0.14 ng BEE and cholic acid:
8,602 cpm (B), or a B/Bo of ?1% (indicating presence of BEE) The similarity of the resulting-B/Bo's (76% and 71%) with and without cholic acid indicate that cholic acid did not interfere with the assay for cocaine or benzoylecgonine.
Examt~Ie I2 Digestion of Hair Using Proteinase K and Removal of Interfering.Substance In Marijuana Assay.
ZO mg of hair from a known no ..al subject (not marijuana users) and 10 mg of hair from I known marijuana user were placed in separate 13 x ?5 mm polycarbonate tubes. One ml of the following solution was added to the hair in each of the tubes: 0.5M Tris buffer (pH 6.5 at room temperature) containing in one ml 2 U Proteinase K, 2 mg cholic acid (sodium salt), and 60 mg dithiothreitol. The mixtures were shaken 16 hours at 3?' C. after 16 hours, the samples were centrifuged at 3 , 000 Rp~i for 20 minutes . To 0 . 9 ml of supernatant removed after centrifugation was added 10 p.l of phenylmethylsulfonyl flucride (6~ in ethanol). This ;aas mixed, followed by the addition of 90 ul CuSO, . ~iiZO ( 10 ../liter). The mixtures were shaken at 37' C fcr 30 minutes.
3~
SUBSTnUTE SHEET {RULE 26) Six grams of dry A-25-120 DEAE Sephadex (TM) available from Sigma Chemical Co.
of St.
Louis, Missouri, was added to 100 ml of distilled, deionized water. The suspension was allowed to swell overnight in the refrigerator (2-8°C). After swelling, the suspension was placed in a beaker with a large magnetic stirring bar, and was vigorously stirred on a magnetic stirring plate for 60 minutes until the resin had broken up into small particles resulting in a very fine suspension.
To 400 ~,1 of each of the hair digest solutions was added 300 ~,1 of the 6 %
resin suspension in a glass tube. The glass tubes were rotated vigorously (greater than 200 rotations/minute) for 30 minutes in such a way as to keep the resin suspended and in contact with the digest solution.
To each of the tubes was added 0.7 ml distilled water. The tubes were then centrifuged, and 1.0 ml of the supernatant from the resinated digest solutions was removed. 200 ,ul of 1M
phosphate buffer was then added to each solution. 100 ~.1 of the filtrate of each sample was then assayed for marijuana by RIA. The results were as follows:
Sample 1 (known negative) 4597 cpm Sample 2 (known positive) 2683 cpm Sample 2 demonstrated a B/Bo of 59% as compared to negative samples (100%), a positive result for the presence of marijuana analyte in Sample2.
Example 13 Known Negative Hair Samples Compared To Known Positive Hair Samples Using Ion Exchange Resin.

:~~~~~~so Eight negative hair samples known to show great variability in binding in the marijuana assay due to varying amounts of interfering cross reacting substance, and a hair sample from a known marijuana user were digested and neutralized by the procedure described in Example 12. The digests were treated with water suspensions of various resins prepared in the concentrations (as percent of bottled resin relative to total suspension) shown below and the resinated digest solution was then tested in RIA.
In more detail, 0.4 ml of the digest solution was mixed 0.3 ml of the resin suspension in a 13 x 100-mm glass tube, and the tubes were shaken at >200 rpm for 30 minutes. Then 0.75 ml of distilled water was added to each of the tubes, and the tubes were mixed well and centrifuged at 200 R.'~M for 10 minutes. One ml of supernatant was removed from each and placed in a 13 x 100 mm glass tube, In performing the marijuana assay, 200 ~ul of 1 M, pH 7 phosphate buffer was added to the tube, followed by 100 ul It2~ marijuana tracer and 100 ,ul anti-~cannabinoid antibody.
The tubes were mixed and incubated 1 hour at room temperature. 200 ~.al of a second precipitating antibody was added followed by 1 ml 6o polyethylene glycol. After 30 minutes at room temperature, the tubes were centrifuged at 3000 RPM in a re=rigerated centrifuge. After decanting, the ' tubes wer a coun~ed in a gaa~ a sz~ec trometer .

' SUBSfITItfE SHEET RULE 26) the means and standard deviations of the counts of the negatives were calculated to determine the effectiveness of the resins in removing the interfering cross reactor substance. The counts of the positive hair sample digest divided by the mean of negatives was calculated to determine how well the analyte was not removed.
The following results were obtained using a 2.4 % resin suspension of DEAE-Sephadex (TM) (A-50-120):
Sample CPM

Negative # 1 3961 Negative #2 4391 Negative #3 3712 Negative #4 3804 Negative #5 3574 Negative #6 4004 Negative #7 4010 Negative #8 4252 Positive #1 2489 Positive #2 2424 The mean of the negative samples was 3964, with a standard deviation of 238 (6 % of the mean). The B/Bo of the positive samples is 62% . Thus, the signal to noise ratio indicates that the assay is sensitive.
For DEAE Sepharose (TM) (CL-6B), the following results were obtained using a 24 % resin suspension:
Sample CPM

Negative # 1 4085 Negative #2 4070 Negative #3 3661 Negative #4 3771 Negative #5 3555 Negative #6 3677 Negative i7 ~ ~~ ~ ~ ~ 3806 Negative ft8 4137 Positive 1 2550 Positive n2 2539 The mean of the negative samples was 3845, with a standard deviation of 196 (5% of the mean). The B/Bo is 66%. Thus, again, the signal to noise ratio indicates that Z5 the assay is sensitive.
While there have been described what are presently believed to be preferred embodiments of the invention, it >r' will be apparent to one stilled in the art that numeraus changes can be made in the ingredients, conditions and proportions set forth in the foregoing embodiments without departing from the invention as described herein and as defined in the appended claims.

SUB~'r~ltf E SHEET (RULE 26)

Claims (6)

WHAT IS CLAIMED IS:

1. A method for the detection of marijuana intake by an individual which comprises:
(a) chemically treating a sample of hair from the individual in a manner that releases an analyte which has become embedded in the hair from the bloodstream of the subject as a result of marijuana ingestion to form a test solution; said solution containing an interfering cross reacting substance naturally found in hair;
(b) preparing a suspension of an ion exchange resin in a concentration and amount sufficient to remove the interfering substance from the test solution such that the analyte may be detected by immunoassay using an antibody which specifically binds to cannabinoids;
(c) mixing the test solution and sufficient suspension to remove the interfering substance from the test solution; and (d) after the interfering substance has been substantially removed from the test solution, subjecting a portion of the solution to analysis by immunoassay using an antibody which specifically binds to cannabinoids to detect the analyte if present.

2. A method for the detection of marijuana intake by an individual which comprises:
(a) preparing a mixture comprising an agent selected from the group consisting of dithiothreitol and dithioerythritol, proteinase K suitable for the digestion of hair, and a sample of hair; the amounts of the proteinase K and the agent being sufficient to effectuate the digestion of the sample;
(b) permitting the digestion of the sample of hair sufficient to release an analyte which has become embedded in the hair from the bloodstream of the subject as a result of marijuana ingestion to form a test solution; said solution containing a cross reacting interfering substance naturally found in hair;
(c) permitting the passage of a sufficient period of time for the agent to become deactivated such that said agent will not interfere with the accuracy of an immunoassay method performed on the test solution;
(d) preparing a suspension of ion exchange resin in an amount and concentration sufficient to remove the interfering substance from the test solution;
(e) mixing the test solution and the suspension to remove the interfering substance; and (f) after the agent has deactivated, and the interfering substance has been substantially removed, subjecting a portion of the test solution to analysis by an immunoassay using an antibody which specifically binds to cannabinoids to detect the analyte if present.

3. The method according to Claim 2 wherein the hair sample is digested at a pH
between about 7 and about 9 at a temperature between about 20 and about 40 degrees centigrade.

4. The method according to Claim 2 further comprising the step of adding a compound that produces copper ions in solution to the test solution after the hair sample has been digested and before the test solution is subjected to analysis by immunoassay in an amount sufficient to deactivate the agent such that the time necessary for the deactivation of the agent is decreased.

5. A method for the detection of marijuana intake by an individual which comprises:
(a) chemically treating a sample of hair from the individual in a manner that releases an analyte which has become embedded in the hair from the bloodstream of the subject as a result of marijuana ingestion to form a test solution; said solution containing a cross reacting interfering substance naturally found in hair;
(b) preparing a fine suspension of slowly settling particles of anion exchangers on dextrose in an amount and concentration sufficient to remove the interfering substance from the test solution such that the analyte may be detected by immunoassay using an antibody which specifically binds to cannabinoids;
(c) mixing the digest solution and the suspension to remove the interfering substance from the test solution; and (d) after the interfering substance has been substantially removed from the test solution, subjecting a portion of the test solution to analysis by immunoassay using an antibody which specifically binds to cannabinoids to detect the analyte if present.

6. A method for the detection of marijuana intake by an individual which comprises:
(a) chemically treating a sample of hair from the individual in a manner that releases an analyte which has become embedded in the hair from the bloodstream of the subject as a result of marijuana ingesting to form a test solution; said solution containing a cross reacting interfering substance naturally found in hair;
(b) preparing a suspension of slowly settling particles of ion exchange resins selected from the group consisting of anion exchangers on dextrose; anion exchangers on agarose; anion exchangers on cellulose; cation exchangers on dextran; strongly acidic cation exchangers on polystyrene; benzyl diethylaminoethyl on cellulose; and triethylaminocellulose in an amount and concentration sufficient to remove the interfering substance from the test solution such that the analyte may be detected by immunoassay using an antibody which specifically binds to cannabinoids;
(c) mixing the test solution and the suspension to remove the interfering substance from the test solution; and (d) subjecting a portion of the hair digest solution to direct analysis by immunoassay using an antibody which specifically binds to cannabinoids to detect the presence of the analyte.