(12) Patent Application Publication (10) Pub. No.: US 2015/0017732 A1 Wu Et Al

Home , Dimethoxymethamphetamine

US 2015.0017732A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0017732 A1 Wu et al. (43) Pub. Date: Jan. 15, 2015

(54) COLORIMETRIC METHOD TO DETECT Publication Classification ILLICT DRUGS (51) Int. Cl. (71) Applicants: Tsunghsueh Wu, Platteville, WI (US); GOIN33/94 (2006.01) Charles Cornett, Platteville, WI (US) GOIN 2L/78 (2006.01) (52) U.S. Cl. (72) Inventors: Tsunghsueh Wu, Platteville, WI (US); CPC ...... G0IN33/94 (2013.01); G0IN33/946 Charles Cornett, Platteville, WI (US) (2013.01); G0IN33/948 (2013.01); G0IN 33/9486 (2013.01); G0IN 21/78 (2013.01) (21) Appl. No.: 14/329,714 USPC .. 436/92: 436/96; 436/98; 422/430; 436/111; (22) Filed: Jul. 11, 2014 436/93 Related U.S. Application Data (57) ABSTRACT (60) Provisional application No. 61/937,356, filed on Feb. 7, 2014, provisional application No. 61/845,632, filed A colorimetric assay to detect certain compounds and a kit on Jul. 12, 2013. therefore are provided.

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SOd

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}} } } } } } } } } } } } } } } } } } } } } } } } } } } } } } }} } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } 69 Patent Application Publication Jan. 15, 2015 Sheet 21 of 67 US 2015/0017732 A1

SOd uMoug

SOd

8.au

sod

?a?isod 8.au

0A. ?A. Patent Application Publication Jan. 15, 2015 Sheet 22 of 67 US 2015/0017732 A1

Patent Application Publication Jan. 15, 2015 Sheet 24 of 67 US 2015/0017732 A1

Patent Application Publication Jan. 15, 2015 Sheet 25 of 67 US 2015/0017732 A1

SOd SOd

83N SOd SOd Patent Application Publication Jan. 15, 2015 Sheet 26 of 67 US 2015/0017732 A1

Patent Application Publication Jan. 15, 2015 Sheet 28 of 67 US 2015/0017732 A1

Patent Application Publication Jan. 15, 2015 Sheet 29 of 67 US 2015/0017732 A1

SŠ y S S Y= S

S S S S S S S S SŠ

S S S S &

S &

Patent Application Publication Jan. 15, 2015 Sheet 31 of 67 US 2015/0017732 A1

& 8&

8 & 8 & 8

8 S Patent Application Publication Jan. 15, 2015 Sheet 32 of 67 US 2015/0017732 A1

Patent Application Publication Jan. 15, 2015 Sheet 36 of 67 US 2015/0017732 A1

Patent Application Publication Jan. 15, 2015 Sheet 37 of 67 US 2015/0017732 A1

Drug ph 2 ph A. pH 7 Structure Photograph

Control | Negative Negative Negative

Negative Negative Negative ------s ------A. l s Histidine N- OH & is Cas: 23B-230) Š

White Powder 2 Negative Negative Negative & 8 s

Arginine ---^& 3.1. S Cas:36B-228O Š

White Powder

3 Negative Positive Negative Thyrosine Weight H yetiaw: N CaS 6469 - Š

White Powder

A. Negative Negative Negative Dihydroxy pheny -o alarine s y-sa Cas: 59-92-7 Ho Y O White Powder

5 Negative Negative Negative Š Tryptophan

Cas:4903 X----

FIG. 9A Patent Application Publication Jan. 15, 2015 Sheet 38 of 67 US 2015/0017732 A1

White Powder 6 Negative Negative Negative

Threonine

CAS:W853

White Powder

7 Negative Negative Negative Cysteine

CAS7439

White Powder ------8TT Negative Negative Negative --- Aspartic Acid ---, CAS. 4873 sy N is White Powder

9 Negative Negative Negative

Phenylalanine &- CAS93 N- s

White Powder 1. positive 1.-Methylindole N CAS: 63-76-9 A Brown Liquid Patent Application Publication Jan. 15, 2015 Sheet 39 of 67 US 2015/0017732 A1

C I-Methylindole-3- very sight 3. N :-

carboxylic acid yellow s

s White Powder Cis 13 Negative is g is s s &- WH2OO - -, -?i-. r X. CAS 13 &--

Clear liquid 1. Negative

WHO38

CAS: 13369

Yellow liquid

15 Negative

WHO.5

CAS: 4368-23

White powder

16 Rositive Endoire

CAS:496-5- light Brown liquid

1. C3 Trethylamine C -- \ S-Chis CAS: 21-44-8 Yellow liquid

FIG. 9C Patent Application Publication Jan. 15, 2015 Sheet 40 of 67 US 2015/0017732 A1

18 Negative positive Negative Tetramethyl-amminium

hydroxide

CAS:504952

White powder

19 Negative Negative

1,2-Dimethylindoi ty sight yellow: CAS: 875-79-6

Brown Powder

O Negative positive Negative

Thiourea very light

yellow CAS 2938

White powder

2 Positive Basitive Methysergide Maleate Eight

yellow CAS:32O. White powder

s 22 Negative Negative Negative 2-Tichloromethyl-4-3h)- quinazolinone

CAS; S558-95-2

White powder

23 Negative Negative Negative

6-methoxy-4-methyl

Coimarin

FIG. 9D Patent Application Publication Jan. 15, 2015 Sheet 41 of 67 US 2015/0017732 A1

CAS 5295-35-3 White powder

24 Negative Tetramethylaimonium chloride

CASW635 White powder

25 Ouinine Sulfate

CAS: 619-JO-6 White powder 26 Negative Negative Negative Shioacetamide Cloudy

CAS:XO475-3 White powder 27 Negative 1(Phenylthio)methyl)- iH-benzotriazole

CAS:1198-03-9 White powder 28 Negative Negative Negative Naphthalere

CAS:58 White powder

Not water Soube

FIG. 9E Patent Application Publication Jan. 15, 2015 Sheet 42 of 67 US 2015/0017732 A1

29 Negative Negative

1H-Benzotrialoe

CAS:95-14-7 White powder

3O so-Butylamine

CAS: 2508 Yellow liquid

33 Dibenzylamine

CAS: 103-9- Light yellow liquid

32 Negative Negative Negative Ampicillin

CAS835.242 White powder

33 Negative Negative Negative

warnto

M-120 White powder

34 rekiössists

. . . 3 cities.ch Trihexydiethylene triamine

CAS; 258-15-6

FIG. 9F Patent Application Publication Jan. 15, 2015 Sheet 43 of 67 US 2015/0017732 A1

Light yellow liquid

35 Negative Negative Negative

Caffeine

CASS8-08-2 White powder

& Bositive Dexto Methophan Hydrobromide

CAS:125-69-9 White powder 37 PiSiiga his -*s-C Chlorpheniamine cr'Y- - - N- Maleate -is CAS; 903.25 a White powder 38 Negative Negative Negative

Boric--. acidPrs - --8-{-

CAS: White powder YYYYYYYY-MMYYYgrilNegative Negative T Negative Tir TT rst T g g HOS-r-OH Resorcino: % CAS: 3380 White powder

FIG. 9G Patent Application Publication Jan. 15, 2015 Sheet 44 of 67 US 2015/0017732 A1

40 Negative -- Na" "OOH Baking Soda i CAS: White powder

4. Negative

Advi White powder with ed Coating A2 Negative Negative Negative: Adult Strength Pain Reliever White powder

43 Negative Negative Negative ibuprofen White powder

44 Negative Tylenol Extra Strength White powder with blue and red Coating ------Positive Benadry White powder

A5 Negative

Motrir White powder

FIG. 9H Patent Application Publication Jan. 15, 2015 Sheet 45 of 67 US 2015/0017732 A1

47 Positive: 1-Methylindole-3- Siery light carboxaldehyde 3e3

CAS;902-03-4 White powder

3-(3-Hydroxypropyl)-1H indole

CAS: 3569-2-9 thick yellow figuid

A9 Negative Negative Negative C-: 8. O inositol

AS: 87-898 C White powder -: SO positivisiósitive: Positive S rus ::::::::::::: r Biphenhydrainine Ye ?h, hydrochloride 1s. or -'ch, . He & CAS; 147-24-0 sa White powder

S. Positive N-(2-Hydroxyethyl pyro CyY lidine N CAS: 2955-38-6 Yellow liquid N-OH Patent Application Publication Jan. 15, 2015 Sheet 46 of 67 US 2015/0017732 A1

5,7-Dimethylisatir Orage

CAS3968-2A

Red powder

53 Östie

ridodecyjamine

CAS102-37-4

Colorless liquid

54 isities Six isiis

Procaine

CAS: 59-45-1 White powder

idocaine

CAS;137-53-6 White powder

56 Negative Negative Negative Dimethyl Suifone

67-71 White powder

57 8-Methoxyiuloidine

CAS:44-3-3 White powder

FIG. 9 Patent Application Publication Jan. 15, 2015 Sheet 47 of 67 US 2015/0017732 A1

58 Negative Negative Negative O

Benzoylnaphthalene I r s SS CAS:63468-83-7 Š-Y3 S- & Brown liquid S9 Negative POSitive Negative

Acetamide

CAS: White powder2

60 Negative Negative Negative Piperonal

CAS:1032.432 White powder 6. Negative Negative Negative

Capsaicin

CAS.404-86-4 White powder 62 Fositive Xtra detergent Topsid

bottorn, , , White powder yet. cictly: -- Negative

Cascade Bottom contains White powder soid

FIG. 9K Patent Application Publication Jan. 15, 2015 Sheet 48 of 67 US 2015/0017732 A1

64. : Risitive OX: Cear Siery tight yellow White powder

Ketanine Colorless liquid

66 Site

Hero Colorless liquid

|-|--|--6 R8sitive : : Cocaire Colorless liquid 68 Negative

Tea extract Brown liquid TT Negative Negative Negative Carbamazepine

7. Rositive::::: Positivie Positive (t)-Metoprolo

FIG. 9L. Patent Application Publication Jan. 15, 2015 Sheet 49 of 67 US 2015/0017732 A1

7. Negative Negative Negative 3-5-(triflouromethyl)-2- pyrid piperidine-4- carboxylic acid

72 Negative Negative Negative Sugar White powder

73 Sweetener sugar substitute White powder

74 Negative Negative Negative Non-dairy creamer Light orange White powder cloudy top layer

75 :::::::::::: YY ---, -i-. SD - & N Colorless liquid i.e. S

f : Methamphetamine S r CH Colorless liquid

FIG. 9M Patent Application Publication Jan. 15, 2015 Sheet 50 of 67 US 2015/0017732 A1

77 Positive

MDMA Colorless liquid

Burnelidocaine)

White clearn

79 posit ive: ora anesthetic paste (benzocaine) yellow paste 8O Positive O Albeso (benzocaine) Red liquid

Negative

EAM.220 Colorless liquid

FIG. 9N Patent Application Publication Jan. 15, 2015 Sheet 51 of 67 US 2015/0017732 A1

82 Negative 1'-Naphthoylindole Colorless liquid

83 positive

VDPA x -: Colorless iduid

84 :: Positive 3,4- Dimethoxymethampheta nine Colorless liquid 85 Positive 2,3- dinnethylethcathinoe

Wincamine White powder

4-fluoromethcatione

2 methoxymethcathinone

FIG. 90 Patent Application Publication Jan. 15, 2015 Sheet 52 of 67 US 2015/0017732 A1

9. ^ -: - - ‘S. ' ^s- Methadone - &. s: --- 8 N

s: 93 i ^ re -: s Butylone -s-s -- \- w

94 3-rnethylbuphedrone

95. 3,4-dimethylcathinone

96 3,4'-methylenedioxy-o- pyrrolidinopropiopheno e

97 8: 3-fluoromethcathinoned ---.S.---- i. iii.

FIG. 9P Patent Application Publication Jan. 15, 2015 Sheet 53 Of 67 US 2015/0017732 A1

101 negative

Aprazolan

O2 negative

Clonazepam

O3 negative H.y

SS Diaean 1-: stiy d

w; Y,f Sea

104 negative R. o

Fiumitrazepam

OS negative

Orazepam

FIG. 9C) Patent Application Publication Jan. 15, 2015 Sheet 54 of 67 US 2015/0017732 A1

negative Ditrazepam

negative Oxazepan

negative Temazepam

2-Amino-4- bromoacetophenone hydrochloride

322 2',4'- dimethylacetophenone Chi,

FG. 9R Patent Application Publication Jan. 15, 2015 Sheet 55 Of 67 US 2015/0017732 A1

23 2-aminoacetophenone hydrochloride

24 -- - o 2-Amino-4'- r Niki methoxyacetophenone iQo & e is hydrochloride va

25 O 4'-chloroacetophenone ?cr's C

26 C 3'-chloroacetophenone C s Šs 27 -- - O 2'-chloroacetophenone l 21\c 50 -:

- C ..i -OH ci Ya- N ii- y - l Patent Application Publication Jan. 15, 2015 Sheet 56 of 67 US 2015/0017732 A1

r 151. : " o 4-(dimethylamino)- a- - - i-xy Benzoic acid : s

rŠs

st:3: 3.38.

153

159

o-chloro-p- : fluoroacetophenone

6. p-chloropherol

62 dibromoacetophenone2,4'- -s-\-- ---

FIG. 9T Patent Application Publication Jan. 15, 2015 Sheet 57 of 67 US 2015/0017732 A1

163 T. 2,4-dichlorobenzoic acid ls s Sc. - 2e.

------1.65 p-nitrobenzoic acid Cl

300 Negative Negative Negative {t}-amphetamine

30 Negative Positive Positive 6-Acetymorphine

--- 3. 3O2 Positive Rositive Positive s 8-acetylcodeine

303 Negative Negative Negative

Androstenedione

FIG. 9U Patent Application Publication Jan. 15, 2015 Sheet 58 of 67 US 2015/0017732 A1

"30 Negative Negative Negative C Butaita his Al \ Ql - s 3 -- -N

305 Negative Negative - Megative

& & Gamma-butyrolactone : & Š

306 Negative Negative Negative

butabarbital

307 Negative Positive Negative 4-bromo-2,5- dimethoxyphenethylami e 3O8. Negative positive positive Codeine

309 Negative Positive negative

Cocaine

FIG. 9V Patent Application Publication Jan. 15, 2015 Sheet 59 of 67 US 2015/0017732 A1

330 Negative Negative Negative

Cathinone (50 ug (200 ug (50 ug

3. Negative Negative Negative

Cannabidio

38.2 Negative Rositive Negative 4-chloro-2,5- dimethoxyphenethylami

333 Negative :: Positive: Rositive

1S,2R(1) Ephedrine hydrochioride

334 Negative Negative Negative (t)-N-ethylamphetamine

335 88sitive. Eositive. asitive Hydrocodone

FIG. 9W Patent Application Publication Jan. 15, 2015 Sheet 60 of 67 US 2015/0017732 A1

hydromorphone

317 Positive Positive: Positive Heroir

3.18 positive positive negative S::

Ketamine S. $

319 Positive: Positive

320 Negative Negative Negative Morphine

32. Negative Negative Negative Methamphetamine

------Patent Application Publication Jan. 15, 2015 Sheet 61 of 67 US 2015/0017732 A1

322 Eositive: isitives: 88sitive

323" |Negative Negative Negative Methaqualone

------"T" Negative Negative Methylephedrine

325 Negative sists Sive

Dorcodine

326 Negative Oxycodone

327 Positive positive Positive PCP

323 Negative Negative Positive

Penterrine

FIG. 9Y Patent Application Publication Jan. 15, 2015 Sheet 62 of 67 US 2015/0017732 A1

329 Negative Negative Negative

330 Negative Negative Negative

R-44

33 positive Negative

AV 248

332 Negative Negative Negative (-)-deltaS-THC

333 Negative Negative Negative

testosterCa Patent Application Publication Jan. 15, 2015 Sheet 63 of 67 US 2015/0017732 A1

2,3- \* -S.s s \ --- dimethylethcathinone --

90 Positive Fasitive 4-flooreticatione {:g):::::::g

sees 91 ::::::::::::::::Rositive Sitive Eastes 2- 3rag nethoxymethcathinone

93 Positive: Fasitive Butylone {x} ing) (ing)

S4 Fositive ositive

3-methylbuphedrone {}ing...::::: (: Eng)

3,4-dimethylcathinone &ing Patent Application Publication Jan. 15, 2015 Sheet 64 of 67 US 2015/0017732 A1

96. Pasitive positive asitive . - o:

3',4'-methylenedioxy-q- (xiiig. (3 tig pyrroidinopropopheno e

97 3-fluoronethcathinone Y-s -- t -Yss&

FIG. 9BB Patent Application Publication Jan. 15, 2015 Sheet 65 of 67 US 2015/0017732 A1

MO/Scott's Reagent

To be determined (TBD)

37 50

327

11 1-Methylindole-3-carboxylic acid - - colorless 83 MDPA - TBD 29 H-Benzotriazole - - TBD 334 3,4-Dimethoxymethamphetamine - TBD soo amphetamine red 301 G-Acetylmorphine TBD 302 6-acetylcodeine - TBD 303 Androstened one BD Butabital TBD 30S Gamma-butyrolactone outabarbita 307 4-bromo-2,5-dimethoxyphenethylamine 31 Cannabidio 32 4-chloro-2,5-dimethoxyphenethylamine T BB 34. (t)-N-ethylamphetamine 35 Hydrocodone 36 hydromorphone T B 37 heroin ET B 39 S 32 Morphine 3. 21. BD 322 MDA - I - TBD 323 Methaqualone - BD 324 Methylephedrine - - TBD

FIG. 10A Patent Application Publication Jan. 15, 2015 Sheet 66 of 67 US 2015/0017732 A1

325 | Norcodine TBD 326 oxycodone - TBD 328 Phentermine - TBD 329 XLRI. TBD R-14 TSD AM 1248 TB)

(-)-deta9-THC - T - TBD 333 testosterone - TBD Note: Al 300 numbers are controlled substances All 90 numbers are synthetic cathirones All tests have been carried out in a Sunny day and simulated night time.

FIG. 1 OB Patent Application Publication Jan. 15, 2015 Sheet 67 of 67 US 2015/0017732 A1

Œil|sdorpgTitz||Eulepool US 2015/0017732 A1 Jan. 15, 2015

COLORMETRIC METHOD TO DETECT fobenzenesulfonate sodium salt. In one embodiment, the ILLICT DRUGS buffer has a pH of about 0 to 8, e.g., 2 to 8. In one embodiment, the buffer has a pH of about 4, e.g., a pH that is from 3.5 to 4.5. CROSS-REFERENCE TO RELATED In one embodiment, the buffer is a phosphate, Sodium acetate, APPLICATIONS citrate, phosphate-citrate, tartrate, oracetic acid buffer. In one 0001. This application claims the benefit of the filing date embodiment, the mixture is compared to a negative control, of U.S. application Ser. No. 61/937,356, filed on Feb. 7, 2014, e.g., which has the buffer, the organic solvent and the dye but and U.S. application Ser. No. 61/845,632, filed on Jul. 12, lacks a piperazine, indoline, indole, azabicyclo, morphinan, 2013, the disclosures of which are incorporated by reference or amine containing compound, and/or a positive control herein. sample, Such as one having the buffer, the organic solvent, the dye and at least one of a piperazine, indoline, indole, azabi BACKGROUND cyclo, morphinan, or amine containing compound. In one embodiment, after combining the test sample and the buffer, 0002 Color tests have been used by criminalists for organic solvent and dye, the organic phase is yellow, blue, decades to provide a rapid, inexpensive means of determining pink, or red. In one embodiment the positive control sample if an unknown compound merits further investigation. Law includes at least one of a benzylpiperazine (BZP), trifluorom enforcement officers rely upon commercially produced, pre ethyl phenylpiperazine (TFMPP), methamphetamine, 3-me sumptive color tests to determine probable cause for arrest thylenedioxy-methamphetamine (MDMA), cocaine, a syn and Subsequent Substance identification by crime laboratory thetic cathinone, or a synthetic cannabinoid. In one personnel and techniques. Following a positive field pre embodiment, the buffer, organic solvent and dye are in a Sumptive test, the evidence (a potential controlled Substance) single receptacle prior to contact with the test sample. In one is sent to the crime laboratory for further processing. This embodiment, the receptacle is formed of a synthetic material. involves an indicative test and finally a confirmatory test. In one embodiment, the receptacle is formed of a plastic. In Color tests may also be used by crime laboratory personnel in one embodiment, the receptacle is formed of glass. In one certain applications, however, the confirmatory test is an embodiment, the receptacle is formed of a translucent mate instrumental assay, typically by gas chromatograph-mass rial. In one embodiment, the first receptacle is present in or spectrometry. can be placed in a larger receptacle that has a mechanism for sealing. In one embodiment, the first or the larger receptacle SUMMARY OF THE INVENTION has a white background for visualization of results. In one 0003. The invention provides a colorimetric assay to embodiment, the composition or reagents have a Volume less detect a piperazine, an indoline, an indole, an azabicyclo, a than 3 mL. In one embodiment, the composition or reagents morphinan, or an amine containing compound. The assay have a volume greater than about 0.5 mL. includes providing a test sample Suspected of having a pip 0004 Also provided is a kit comprising a first receptacle erazine, indoline, indole, azabicyclo, morphinan, or amine having a composition comprising a buffer, an organic solvent, containing compound including but not limited to synthetic and a Sulfonic acid, Sultone or oXothian-2.2.dioxide contain cathinones and synthetic cannabinoids, and reagents includ ing dye. In one embodiment, the dye is methyl orange, Xyle ing an aqueous buffer, an organic solvent, and a sulfonic acid, nol orange, calmagite, metanil yellow, 1-(2-hydroxyl-1- Sultone or OXothian-2.2.dioxide containing dye and option naphthylazo)-2-naphthol-4-Sulfonic acid Zinc salt, 1-(2- ally a Dragendorff's reagent. The reagents may be provided in hydroxyl-1-naphthylazo)-2-naphthol-4-Sulfonic acid Zinc a single receptacle, e.g., an ampoule, or in individual recep salt, 3-((E)-(4-((E)-(4-amino-7-sulfonatonaphthalen-1-yl) tacles which are combined, to form a composition having the diazenyl)-7-sulfonatonaphthalen-1-yl)gliaZenyl)naphtha buffer, the organic solvent and the dye. The sample and the lene-1,5-disulfonate Sodium salt, 2-((4-hydroxyphenyl)(4- reagents are combined and optionally agitated to provide a oxocyclohexa-2,5-dien-1-ylidene)methyl)benzenesulfonate mixture. Then it is determined whether the mixture with the sodium salt, or 4-((E)-(4-(ethylamino)-3-methylphenyl)((E)- test sample has a different color than a control mixture that 4-(ethyliminio)cyclohexa-2,5-dien-1-ylidene)methyl)-3-sul lacks the test sample. In one embodiment, the test sample is a fobenzenesulfonate sodium salt. In one embodiment, the kit Solid, e.g., in the form of a powder. In one embodiment, the further comprises one or more other receptacles, e.g., a sec test sample is in liquid form. In one embodiment, if the test ond receptacle having a composition comprising the buffer, sample has a different color, the intensity of the color may the organic solvent, a Sulfonic acid, Sultone or OXothian-2.2, detected. In one embodiment, the Dragendorff's reagent, e.g., dioxide containing dye, a dye other than a Sulfonic acid, a reagent containing a mixture of bismuth nitrate, glacial Sultone or OXothian-2.2.dioxide containing dye or other test acetic acid potassium iodide and water. In one embodiment, reagent, e.g., the Marquis test or Scott's reagent. In one the dye is a synthetic dye, e.g., one for laboratory use or a food embodiment, the buffer has a pH of about 2 to 5. In one grade dye. In one embodiment, the dye is a natural dye, e.g., embodiment, the buffer has a pH of about 0 to 8, e.g., 2 to 7 or one for laboratory use or a food grade dye. In one embodi 8. In one embodiment, the buffer is a phosphate, sodium ment, the dye is methyl orange, Xylenol orange, calmagite, acetate, citrate, phosphate-citrate or acetic acid buffer. In one FD&C blue 1, metanil yellow, 1-(2-hydroxyl-1-naphthy embodiment, the receptacle is formed of a synthetic material. lazo)-2-naphthol-4-sulfonic acid Zinc salt, 3-((E)-(4-((E)-(4- In one embodiment, the receptacle is formed of a plastic. In amino-7-sulfonatonaphthalen-1-yl)gliaZenyl)-7-sul one embodiment, the receptacle is formed of a glass. In one fonatonaphthalen-1-yl)diaZenyl)naphthalene-1,5- embodiment, the organic solventis chloroform, toluene, dim disulfonate sodium salt, 2-((4-hydroxyphenyl)(4- ethylformamide or dimethylsulfoxide. In one embodiment, oxocyclohexa-2,5-dien-1-ylidene)methyl)benzenesulfonate the organic solvent is chloroform, toluene or Xylene. In one sodium salt, or 4-((E)-(4-(ethylamino)-3-methylphenyl)((E)- embodiment, the buffer is a phosphate, sodium, citrate, tar 4-(ethyliminio)cyclohexa-2,5-dien-1-ylidene)methyl)-3-sul trate, phosphate-citrate or acetic acid buffer. The receptacle US 2015/0017732 A1 Jan. 15, 2015

may beformed of a material that is resistant to degradation by 0014 FIGS. 7A-E. The results of a test for 4 different the reagents. A kit may contain written instructions and synthetic cathinones with methyl orange. optionally a color chart, e.g., to determine the amount of the 0015 FIGS. 8A-E. The results of a test for 4 different compound that is detected. synthetic cathinones with Dragendorff's reagent. 0005. The dyes useful in the methods or kits of the inven (0016 FIGS. 9A-BB. Results for different drugs tested at tion are present in amounts useful to detect the compounds in pH2, pH4 or pH 7. amounts from about 0.001 mg to about 30 mg, e.g., from (0017 FIGS. 10A-B. Results with two reagents including about 0.001 mg to about 0.01 mg, about 0.01 mg to about 0.1 Scott's test (cobalt thiocyanate). mg, about 0.1 mg to about 20 mg, about 1 mg to about 3 mg (0018 FIG. 11. Results with cocaine and Benadryl. and up to about 10 mg. The kits may be used under a variety Benadryl (50) and cocaine (309) were subject to the same of environmental conditions, including at temperatures from reagents and conditions and the color was recorded after each about -3°C. to about 40° C. addition. The end result is the modified Scott's test. Cocaine 0006 For example, a test sample is added to one compart and benadryl show different final results, and so these ment in a receptacle having two compartments, where the reagents differentiate between the two substances. second compartment has the reagents described herein. Then the receptacle is Subjected to pressure to mix the components DETAILED DESCRIPTION in the two compartments. 0019 Color tests have been used in forensic analysis for 0007 Also provided is a series of assays or kits for sequen decades to preliminarily Screen samples for drugs of abuse. tial screening, e.g., in the same receptacle. In one embodi When combined with certain chemical reagents, many Sub ment, the kit further comprises an additional receptacle hav stances produce a clear, distinct and predictable color. The ing one or more reagents to conduct an additional color reactions are typically not specific to one compound, colorimetric test, e.g., to eliminate or reduce false positives. but are produced by many compounds within a drug class or The kit may thus include reagents for any of the following by unrelated Substances containing a common functional or tests/reagents: Liebermann, Marquis, Chen-Kao (Chen), structural group. It is important to note that aspects of color Simon, Duquenois-Levine, Scott, Mecke, iodoform,2,4-dini reactions have never been fully explained due to the frequent trophenylhydrazine, or Schiffs reagent, and/or tests measur occurrence of anomalous responses. Unexpected color ing the presence of azabicyclo groups, morphinan groups, results and the inherent subjectivity of color interpretation amine groups and carbonyl groups. For example, one recept emphasize the presumptive nature of color tests. Their pur able may have methyl orange and another may have a reagent pose is to indicate the possible presence or absence of certain for the Marquis test. The majority of synthetic cathinones or Substances in a sample. If unexpected or ambiguous results naturally occurring cathinones, when mixed with methyl are observed from a color test, the sample should be subjected orange, will result in a yellow color and, when mixed with a to confirmatory instrumental analysis for identification. reagent for the Marquis test, will show no reaction or a yellow 0020 Current color based presumptive drug tests are sum color depending on the molecular structure of the cathinone. marized in Table 1. The tests mentioned in Table 1 have BRIEF DESCRIPTION OF THE FIGURES specificity for the targeted drugs. 0008 FIG. 1. The results of the presumptive test using TABLE 1 methyl orange. The right ampoule contains benzylpiperazine while the left ampoule is a control experiment. Test Chemicals Positive Results 0009 FIG. 2. The results of a presumptive test using 1-(2- Dillie- Cobalt thiocyanate, methanol, Barbiturates: violet hydroxyl-1-naphthylazo)-2-naphthol-4-Sulfonic acid Zinc Koppanyi and isopropylamine salt. The right ampoule is the control experiment (no drug) Simon's Sodium nitroprusside and Methamphetamine: deep Sodium biocarbonate blue and the left ampoule contains drug (BZP). Marquis Formaldehyde and Sulfuric Opium based drugs will 0010 FIG. 3. Detection of synthetic cannabinoids and Acid give a purple color alkaloids. Vial #1 shows the orange precipitate (ppt) forma Amphetamines will turn it Orange/brown color tion after mixing JWHO18 with Dragendorff's reagent. Vial Mecke Selenous acid and Sulfuric Ectasy (MDMA) will #2 is the Dragendorff's reagent in water where no precipitate acid give a purple color is present. Vial #3 is the Dragendorff's reagent in methanol Heroin will give a (showing no precipitate). Vial #4 shows the ppt formation green color after after adding JWHO18 to Dragendorff's reagent-modified prolonged agitation Scotts Cobalt thiocynate, distilled Cocaine will turn the methyl orange test (DMO test). Vial #5 shows the result of the Test water, glycerin, hydrochloric liquid blue control DMO test when no JWHO18 is present. acid and chloroform 0011 FIG. 4. Depiction of an exemplary kit having a Duquenois- Vanillin, acetaldehyde, ethyl Marijuana will turn Levine alcohol, and chloroform the Solution purple pouch that contains instructions and one or more receptacles PDMAB P-dimethylaminobenzldeyde, LSD will turn blue having a composition comprising a dye. hydrochloric acid and ethyl purple 0012 FIG.5. Depiction of exemplary receptacles having a alcohol composition comprising a dye and a color code for determin Cobalt Acetate Cobalt acetate, methanol and Barbiturates will turn ing whether a test sample has reacted or interacted with the isopropylamine violet-blue dye. 0013 FIGS. 6A-W. Summary of results for exemplary test 0021. In contrast, the colorimetric assay disclosed herein compounds. The concentration of dye used was 0.1% in aque can provide a single presumptive test for a wide variety of ous solution. The concentration of bismuth nitrate was 2 mM drugs. Thus, an investigator needs just one test instead of and the concentration of potassium iodide was 0.15 M in multiple tests to determine probable cause for an arrest for a Dragendorff's reagent. potential controlled Substance. The assay is thus a rapid color US 2015/0017732 A1 Jan. 15, 2015

test for use in the field. For example, an investigator can place TABLE 2 the questioned substance in a disposable ampoule of chemical reagents necessary for the presumptive identification of con Substances that may be used as diluents. trolled substances including but not limited to benzylpipera Acetaminophan Aspirin Baking Soda Benzocaine zine (BZP), 3-trifluoromethyl-phenyl-piperaine (TFMPP), Boric Acid Caffeine Chlorphenir- Dextrome methamphetamine (Meth), MDMA (Ectasy), cocaine, a syn amine thorphan Dimethylsulfone Diphenhydramine Ephedrine Lidocaine thetic cathinone, and/or synthetic cannabinoids. Inositol Mannitol Ibuprofen Procaine 0022. The detection is likely based on the ion-pairing abil Quinine Tea extract BenZphetamine Methysergide ity and extractability of Sulfonic acid, Sultone or oXothian-2, 1'-napthol indole Parmesan Cheese Sweetener Powdered Milk 2.dioxide containing dyes, such as methyl orange, Xylenol Pseudoephedrine Tetracaine' Sucrose Glucose orange, calmagite, metanil yellow, and 1-(2-hydroxyl-1- *Indicates false positive result with methyl orange test naphthylazo)-2-napthol-4-Sulfonic acid Zinc salt, with the controlled Substance at an acidic pH, e.g., a pH of less than 0027. In one embodiment, the assay detects the presence 7.0, including a pH of about 1, about 2, about 3, about 4, about 5, or about 6. In one embodiment, the assay includes placing of synthetic canninoids, e.g., those in Table 3. about 10 milligrams of a Suspected controlled Substance (which may be present with other Substances) in an ampoule TABLE 3 (receptacle) containing about 1 mL of 0.1% dye, about 1 mL Synthetic cannabinoids of pH 4 phosphate buffer, and about 1 mL of chloroform, Controlled substances. Remarks although other volumes, other dye concentrations, other buff Spice products classified as medicinal preparations ers and other organic solvents may be employed. After stir CP-47 ring vigorously, the bottom layer of the liquid in the ampoule 497-C6, C7, C8, C9 will turn yellow for methyl orange dye and red for the 1-(2- H-018 -210 hydroxyl-1-naphthylazo)-2-naphthol-4-Sulfonic acid Zinc H-01S salt if the controlled substance is present (see FIGS. 1 and 2). H-019 H-073 0023. Initially, cetirizine, an anti-histamine, was tested H-2SO with methyl orange. Subsequently, a color change was H-398 detected with the assay when scheduled compounds, e.g., H-200 BZP TFMPP, methamphetamine, or Ecstasy (MDMA), were H-018m H-081 tested. In one embodiment, a powder Sample is added to a test H-122 kit and agitated to mix the reagents with the sample, e.g., for H-098 a few seconds. In the presence of compounds Such as ben -149 Zylpiperazine, MDMA, 2,5-dimethoxy-4-methylamphet H-166 H-175 amine (DOM), or a mixture of synthetic cannabinoids com H-176 pounds, the test Solution changed color from colorless to H-184 yellow. Furthermore, the intensity of the color correlated to H-185 the amounts of the drug that were added. H-192 H-193 0024. The reagents and compositions of the invention H-194 readily detected the presence of heroin, MDMA, metham H-195 phetamine, LSD, JWHO18, synthetic cathinone, and cocaine. H-196 H-197 0025. One advantage of the present assay in the field is that H-198 it is not specific to the scheduled compounds and it is Superior H-199 Leonotis Leontius in identifying azabicyclo, morphinan, amine and indoline Nymphacea caerulea functional groups, which are commonly present in the sched H-398 ule compounds. Existing color test kits (such as those in Table 1) are very specific to the type of scheduled compounds. However, this creates inconvenience for law enforcement 0028. The assay may be employed to detect common officers in analyzing an unknown powder in the field as they scheduled compounds in four classes of compounds, e.g., have to test the same powder in different kits to identify the narcotics, depressants, stimulants, hallucinogens, cannab compounds. In contrast, the test kit described herein can inoids (both naturally occurring and synthetic cannabinoids), detect various scheduled compounds in one test kit, which and cathinones (both naturally occurring and synthetic cathi allows officers to screen a bag of probable drug materials in nones), e.g., Such as 4-methyl-N-methylcathinone (mephe One teSt. drone), 3.4-methylenedioxy-N-methylcathinone (methy 0026. In one embodiment, the assay of the invention lone), and 3.4-methylenedioxypyrovalerone (MDPV). detects a piperazine, indoline, indole, azabicyclo, morphinan, Synthetic cathinones include but are not limited to mephe or amine containing compound even when that compound is drone, methylone, MDPV, butylone, 4-fluoromethcathinone in the presence of other materials, including other scheduled (4-FMC), 3-fluoromethcathinone (3-FMC), 4-methoxymeth compounds and plant materials, which are commonly found cathinone (methedrone), 4-methyl-N-ethylcathinone in the seized samples. Thus, Dragendorff's reagent and dye (4-MEC), ethylone, buphedrone, dimethylcathinone, diethyl reagents disclosed herein useful in one embodiment have cathinone, and 3.4-methylenedioxy-C-pyrrolidinopentiophe little if any reactivity with common Substances, pharmaceu none (MDPBP). “Bath salts' are made from methcathinone tical compounds, and pesticides, such as those shown in Table analogues, typically mephedone and MDPV. Table 4 lists the 2 below. targeted controlled compounds from common scheduled US 2015/0017732 A1 Jan. 15, 2015

compounds in the four classes, representing narcotics, 0029. Dyes useful in the invention have at least one sul depressants, stimulants, hallucinogens, and specific cannab fonic acid, Sultone or oXothian-2.2.dioxide functional group inoids. that may form ion-pairs with compounds including illicit drugs that contain amine (primary, secondary or tertiary), TABLE 4 piperazine (primary, secondary or tertiary), indoline and Class Targeted compound optionally indole groups (which may be present in Synthetic cannabinoids and psilocybins and may be converted to indo Narcotics Opiates line groups) functional groups. A receptacle of the invention Heroin Hydrocodone includes an aqueous layer having the dye at the top and an Morphine organic solvent, e.g., chloroform, layer at the bottom. The test Depressants Barbiturates sample to be added may be in powder or liquid form. In one Benzodiazepines Stimulants Cocaine embodiment, Dragendorff's reagent can be incorporated to Amphetamines detect synthetic cannabinoids and alkalkoids. MDA (3.4-methylenedioxy amphetamine) 0030) Sulfonic acid containing dyes which may be useful MDMA (3.4-methylenedioxy in the methods and kits include those in U.S. Pat. No. 4,560, methamphetamine) 765 and those described in Clanton et al., J. Acquired Immune Hallucinogens Psilocybin Lysergic acid diethylamide (or LSD) Defic. Syndr, 5:771 (1992), the disclosures of which are Phencyclidine incorporated by reference herein. For example, the following Cannabinoids THC dyes may be useful in the assays described herein: Suramin, Synthetic cannabinoids Sulfonic acid-containing azo compounds, 3'-azido-3'- JWHO18 dideoxycytidien (ddC). Chicago sky blue, Evans blue, try AM22O1 Cathinones Cathinones pan blue, direct orange 15, direct blue 15, Erie fast blue, Ink Synthetic cathinones blue, Direct red 75, Erie yellow, and oxathiin carboxyanilide (structures of Some of those dyes, as well as methyl orange and Xylenol orange are shown below).

A: Chicago sky blue MeO OMe

NH2 OH OH NH2 HOS NEN NEN SOH

SOH SOH

B: Ewan's blue Me Me

NH2 OH OH NH2 HOS NEN NEN SOH

SOH SOH

C: Trypan blue Me Me

NH2 OH OH NH2 NEN NEN

HOS SOH HOS SOH

US 2015/0017732 A1 Jan. 15, 2015

0031. The use of color test kits is the most common -continued method for the presumptive identification of drugs in the field because they are quick, easy to use, and cost effective, thus MO D Marquis they are an ideal method for use in a Field Investigative Drug Class Target compound Test Test Test Officer (FIDO) program. Such color test kits provide clear, MDMA (3,4- -- dark unambiguous color to indicate a positive or negative result, methylenedioxy- purple Sufficient specificity to minimize false positive or false nega methamphetamine) Hallucinogens Psilocybin Brown tive interpretation, adequate sensitivity to allow the detection Lysergic acid -- Black of drugs at concentrations commonly encountered in Street diethylamide samples, accurate results for drugs mixed with a variety of (or LSD) adulterants, and reproducible results. Phencyclidine O (or PCP) reaction 0032 To determine if a particular dye, buffer and organic Cannabinoids THC -- Orange Solvent combination is useful to detect and/or quantify a JWHO18 yellow particular compound in a sample, the following test may be AM22O1 conducted. One (1) milligram to ten (10) milligram portions Synthetic Cathinone -- O Cathinones reaction of each sample are weighed. Sensitivity is detected with MDPV -- Yellow samples tested in duplicate. Specificity is detected with 4-methylethcathinone -- yellow samples tested in duplicate. Reproducibility is detected with Methcathinone -- O ten (10) replicates. A single test of unknown samples is con reaction MDMC -- yellow ducted. For example, a one (1), three (3) or ten (10) milligram 4-fluoromethcathinone -- O portion is placed in each test pouch. After about sixty (60) reaction seconds, the hue, value, and/or chroma of the final color for 2 methoxymethcathinone -- O each sample may be recorded, referencing The Munsell Book reaction methedrone -- yellow of Color. butylone -- yellow 0033 For example, two types of ampoules were prepared. 3-methylbuphendrone -- O A methyl orange ampoule was prepared using methyl orange reaction dye and chloroform while a Dragendorff ampoule was pre 3,4-dimethylethcathinone -- O reaction pared using bismuth nitrate and potassium iodide. 3.4 methylendioxy-C- -- -- yellow 0034) For powder samples, approximately 15 mg of the pyrrollidinopiophenone unknown powder is placed into the ampoule and shaken for 3-fluoromethcathinone -- O 30 seconds. The color change is observed for methyl orange reaction 2,3 dimethyl- O-H yellow test, or precipitate formation is observed for the Dragendorff ethcathinone teSt. Steroids methandienone 0035. For liquid samples, approximately 1 mL of liquid is testOSterole Others carisoprodol poured into the ampoule and shaken for 30 seconds. The color diazepam -- change is observed for methyl orange test, or precipitate alprazolam formation is observed for the Dragendorff test. mitragynine -- 0036. For leaf samples, approximately 0.5g of leaves are lorazepam placed into the ampoule and shaken for 30 seconds. The color Y-Hydroxybutyric acid change is observed for methyl orange test, or precipitate formation is observed for the Dragendorff test. 0038 According to the results, all tested synthetic cathi 0037. The results for compounds containing an amine nones showed positive in methyl orange test (meaning color functional group are shown in Table 5. change in the chloroform layer), most of tested synthetic cathinones showed negative in a Dragendorff's test (no pre cipitate), but the third test, the Marquis test, showed mixed MO D Marquis results. The synthetic cathinones produced either no reaction Class Target compound Test Test Test or a yellow color in Marquis test which may be explained by Narcotics Heroin -- -- Purplish two different reactions with the different molecular structures red of the synthetic cathinones. Hydrocodone -- -- Oxycodone -- violet 0039. In summary, the results show that methyl orange test Morphine -- -- purple is very effective in detecting synthetic cathinones (bath salts) Depresants Secobarbital and may be teamed with Dragendorff, an existing test and/or methaqualone -- O reaction a chemical test which can be used to eliminate false positives Phenobarbital in synthetic cathinones detection. Additional tests such as Stimulants Cocaine HCI -- -- O Liebermann, Chen-Kao (Chen), Simon, Duquenois-Levine, reaction Scott, Mecke, iodoform test, 2,4-dinitrophenylhydrazine test, Cocaine Base -- -- O reaction Schiffs reagent, and/or tests measuring the presence of Amphetamine -- reddish amine groups and carbonyl groups (e.g., the iodoform test, Orange 2,4-dinitrophenylhydrazine test, or Schiffs reagent may be Ephedrin -- O employed to detect carbonyl groups), can be employed rather reaction Lisdexamfetamine -- than the Marquis to assist in reducing the rate of false posi Meth -- reddish tives. Orange 0040. In additional testing, it was found that synthetic cathinones have all positive results at three tested pHs (see US 2015/0017732 A1 Jan. 15, 2015

FIG. 9). When the pH increases above pH 7, the yellow 3. The assay of claim 1 further comprising contacting a ion-pair complex will disappear from chloroform layer. A portion of the sample with Dragendorffs reagent or Scott's different dye identified the presence of compound #319 reagent. (LSD),322 (MDA), 331 (AM 1248), and 327 (PCP). A com 4. The assay of claim 1 wherein the composition comprises bination of that dye and methyl orange offers away to narrow a dye other than the Sulfonic acid, Sultone or OXothian-2.2, down the synthetic cathinones. dioxide containing dye. 5. The assay of claim 4 wherein the composition comprises 0041 Further, the reagentina Scotts test may be combined Dragendorff's reagent or Scott's reagent. with methyl orange in one vessel, e.g., an ampoule. That is, 6. The assay of claim3 wherein the Dragendorff's reagent mixing methyl orange in the same test tube (or ampoule) with comprises bismuth nitrate, potassium iodide, glacial acetate Scott's reagent provides a more powerful, single test. A blue acid and water. color is observed when cocaine is present which may elimi 7. The assay of claim 1 wherein the organic solvent is nate several false positives for cocaine found in the current immiscible in water. Scott's test. Synthetic cathinones remain yellow in the mix 8. The assay of claim 1 wherein the buffer has a pH of about ture having methyl orange and Scott's reagent, while many of O to 8. the false positives in the methyl orange-only test turn green. 9. The assay of claim 1 wherein the organic phase of the 0.042 All publications, patents and patent applications are control sample is clear. incorporated herein by reference. While in the foregoing 10. The assay of claim 1 wherein the organic phase of the specification, this invention has been described in relation to test sample is yellow, blue or red. certain preferred embodiments thereof, and many details 11. The assay of claim 1 wherein the dye comprises a have been set forth for purposes of illustration, it will be Sulfonic acid containing dye. apparent to those skilled in the art that the invention is sus 12. The assay of claim 1 wherein the composition is in a ceptible to additional embodiments and that certain of the receptacle formed of a plastic. details herein may be varied considerably without departing 13. The assay of claim 1 wherein the composition is in a from the basic principles of the invention. receptacle formed of glass. What is claimed is: 14. The assay of claim 4 wherein the further dye comprises 1. A colorimetric assay to detect a piperazine, indoline, cobalt thiocyanate. indole, azabicyclo, morphinan, or amine containing com 15. A kit comprising: pound, comprising: a first receptacle having a composition comprising a buffer, providing a test sample Suspected of having a piperazine, an organic Solvent, and a Sulfonic acid, Sultone or an indoline, an indole, azabicyclo, morphinan, or an OXothian-2.2.dioxide containing dye. amine containing compound, and a composition com 16. The kit of claim 15 further comprising a second recep prising reagents including a buffer, an organic solvent tacle comprising a composition having the buffer, the organic and a Sulfonic acid, Sultone or oXothian-2.2.dioxide con solvent and the dye. taining dye; 17. The kit of claim 15 wherein the dye is methyl orange, contacting the sample and the composition so as to provide Xylenol orange, calmagite, metanil yellow or 1-(2-hydroxyl a mixture; and 1-naphthylazo)-2-naphthol-4-Sulfonic acid Zinc salt. detecting whether the mixture has a different color than a 18. The kit of claim 15 wherein the composition further control mixture that lacks the test sample. comprises a dye other than the Sulfonic acid, Sultone or 2. The assay of claim 1 wherein the dye is methyl orange, oXothian-2.2.dioxide containing dye. Xylenol orange, calmagite, FD&C blue 1, metanil yellow, 19. The kit of claim 18 wherein the further dye comprises 1-(2-hydroxyl-1-naphthylazo)-2-naphthol-4-Sulfonic acid cobalt thiocyanate. Zinc salt, or 3-((E)-(4-(E)-(4-amino-7-Sulfonatonaphthalen 20. The kit of claim 15 wherein the buffer has a pH of about 1-yl)gliaZenyl)-7-sulfonatonaphthalen-1-yl)diaZenyl)naph O to 8. thalene-1,5-disulfonate sodium salt.