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PubMed1998

Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III transcription complexes.

In order to optimize the detection of protein-DNA contacts by DNA photoaffinity labeling, we attached four different photoreactive groups to DNA and examined their ability to crosslink yeast RNA polymerase III (Pol III) transcription complexes. Photoreactive nucleotides containing an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB-dUMP) coupled to 5-aminoallyl deoxyuridine were incorporated into the SUP4 tRNATyr gene at bp -3/-2 or +11. Photo-crosslinking with diazirine revealed contacts of Pol III with DNA that are not detected by DNA photoaffinity labeling using an aryl azide, fluorinated aryl azide or benzophenone group attached to DNA. These novel contacts were of the 82 kDa subunit of Pol III with DNA at bp -3/-2 in the initiation complex and of the 82, 40(37) and 31 kDa subunits of Pol III with DNA at bp +11 in elongation complexes stalled at bp +17. These results provide evidence for the subcomplex of the 82, 34 and 31 kDa subunits of Pol III being positioned near the transcription bubble of actively transcribing Pol III, as all three proteins were crosslinked at bp +11 of the stalled transcription complex.

Affinity Labels↗

PubMed2000

Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells.

The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [(125)I]ODN and by photolabelling of living cells with a [(125)I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by the two methods. Its labelling was saturable and competed for by unlabelled ODN of various sequences and irrespective of the presence of a phosphodiester or phosphoro-thioate backbone. This protein remained sedimentable after carbonate extraction, indicating strong membrane association. About half of the total cell amount resisted extensive surface proteolysis, suggesting a dual localisation at the plasma membrane and cytoplasmic vesicles. The protein was purified using a biotinylated ODN-benzophenone conjugate by photocrosslinking followed by streptavidin affinity purification. A sequence obtained by Edman degradation showed no homology with known proteins. Using anti-peptide antisera, labelling by western blotting revealed at 66 kDa a band with comparable properties as found by ligand blotting. Thus, a new membrane protein acting as an ODN receptor has been demonstrated.

Base Sequence↗

PubMed2003

UV filters with antagonistic action at androgen receptors in the MDA-kb2 cell transcriptional-activation assay.

The fact that certain ultraviolet (UV) filters used in cosmetics display estrogenic activity prompted us to study potential actions on androgen receptors (AR) in the human breast carcinoma cell line MDA-kb2, which expresses functional endogenous AR and glucocorticoid receptors (GR) and is stably transfected with a luciferase reporter plasmid. Dihydrotestosterone (DHT), methyltrienolone (R1881), methyltestosterone, danazol, and androstenedione increased luciferase activity, with EC50 values between 0.11 nM (R1881), 0.14 nM (DHT), and 73.5 nM (androstenedione). DHT-induced luciferase gene expression was inhibited by nonsteroidal antiandrogens, hydroxyflutamide, flutamide, bicalutamide, and vinclozolin. In contrast, the steroidal AR agonist/antagonist cyproterone actetate showed agonistic activity in the absence and presence of DHT, which was not blocked by hydroxyflutamide and thus seems not to be mediated by AR. GR-mediated activation of luciferase by dexamethasone was 100 times less potent than DHT and was not antagonized by hydroxyflutamide. The cell line was used for screening of UV filters, benzophenone-3 (Bp-3), benzophenone-4, 3-benzylidene camphor, 4-methylbenzylidene camphor, butyl-methoxy-dibenzoylmethane, homosalate (HMS), octyl-dimethyl-PABA, and octyl-methoxycinnamate. Two of these, Bp-3 and HMS, antagonized DHT-induced AR activation below cytotoxic concentrations, with IC50 of 5.57 10-6 M (HMS) and 4.98 10-6 M (Bp-3). None of the eight UV filters displayed agonistic activity when tested alone, but high concentrations of Bp-3 induced an increase of luciferase activity in the presence of dexamethasone, which was not blocked by hydroxyflutamide or the estrogen antagonist, ICI 182,780. These data indicate that the UV filters Bp-3 and HMS possess antiandrogenic activity in vitro in addition to estrogenic activity.

Arsenic↗

PubMed2004

Contact and photocontact allergy to ketoprofen. The Belgian experience.

Topical ketoprofen (KP) is widely used because of its anti-inflammatory effect. However, photocontact dermatitis is a side-effect. Between May 2001 and June 2002, the Belgian Contact & Environmental Dermatitis Group conducted a prospective, open patch and photopatch test study in 20 patients suspected of KP dermatitis. Severe skin symptoms requiring systemic corticotherapy occurred in 47%. 5 patients were hospitalized. 1 patient showed prolonged photosensitivity. All patients were tested with KP and the other constituents of KP gel. Attribution to KP was demonstrated in all cases. Patch and photopatch tests with KP 2% in petrolatum showed contact photoallergy in 17 patients, contact allergy in 1 patient and photoaggravated contact allergy in 2 patients. 5 patients also reacted to the fragrance components lavender (Lavandula augustifolia) oil and/or neroli (Citrus aurantium dulcis) oil 5% in alcohol. However, in 4 of these, irritant reactions to the ethanolic dilutions could not be ruled out. Additional tests with 3 non-steroidal anti-inflammatory drugs without benzophenone structure ibuprofen, naproxen and diclofenac identified only 1 contact allergic reaction to diclofenac. Cross-reactivity to the substituted benzophenones, oxybenzone and sulisobenzone occurred only to the first in less than 30% of the patients. A high frequency (69%) of contact allergy to fragrance mix was found. Dermatologists should be aware of the severity of photoallergic reactions to KP and the risk of cross-sensitization.

Adolescent↗

PubMed1992

Actinic reticuloid: action spectra and UVA protection factor sunscreens.

This study compares the action spectra on two new patients with actinic reticuloid (AR), with one previously reported patient. All three patients had exquisite photosensitivity in the UVB range being up to 27 times more sensitive than controls. Sensitivity also increased into the UVA Range, all up to 335 nm and, one into the visible. Their photosensitivity made evaluation of the Sun Protection Factor (SPF) of selected, more recent, high-numbered sunscreens easier to compare, because of the shorter time of the minimal erythema dose when tested with a solar simulator. A sunscreen containing butyl methoxydibenzoylmethane (Parsol 1789) with an overall SPF of 16 was not as effective at protecting the actinic reticuloid patients as a higher SPF numbered sunscreen containing a benzophenone (SPF of 40). The Parsol 1789 gave a protection value of 5 in the UVA Range, compared to one of 3 given by the sunscreen containing benzophenone. The SPFs of seven of these commerically available high-numbered sunscreens were compared with controls.

Aged↗

PubMed1993

Determination of threshold UV-A elicitation dose in photopatch testing.

Photopatch testing, although widely used in the diagnosis of photosensitivity disorders, is not standardized. We performed this study to determine the threshold ultraviolet light A (UV-A) dose required to elicit photopatch test responses. 4 patients with previously positive tests were reexposed to the offending allergen, using an incremental dosage regime. Isopropyl dibenzoylmethane (Eusolex 8020), mexenone (benzophenone-10) and oxybenzone (benzophenone-3) produced positive responses at 1.0, 1.0 and 0.7 J/cm2, respectively. Responses to phenothiazines were deemed phototoxic. These results demonstrate that high doses of UV-A (e.g., 10-15 J/cm2) are unnecessary, and that 5 J/cm2 should become the current standard.

Adult↗

PubMed1997

Contact and photocontact sensitivity to sunscreens. Review of a 15-year experience and of the literature.

This review summarizes published and unpublished data of our 15-year experience with sunscreen allergy and photoallergy. From 1981-1996, 402 patients with suspected clinical photosensitivity were patch and photopatch tested with the commercial sunscreens and facial cosmetics that they had used and with chemical UV absorbers, fragrance materials, preservatives, and emollients. 80 patients (20%) (28 men, 52 women) demonstrated allergic and/or photoallergic contact dermatitis to 1 or more UV absorber(s). In 47 patients with photodermatoses or photo-aggravated dermatoses and in 33 subjects with normal photosensitivity, 91 allergic and 84 photoallergic reactions to UV filters were observed. Over the years sunscreens were added to the test series, which since 1989 comprised the following 10 UV absorbers and which induced allergic (a) and photoallergic (pa) reactions (number, type of reaction): 4 UVA absorbers--isopropyldibenzoylmethane (30a/32pa); butyl methoxydibenzoyl-methane (15a/13pa); benzophenone-3 (3a/9pa); benzophenone-4 (0a/0pa); and 6 UVB absorbers--PABA (2a/2pa); octyl dimethyl PABA (1a/2pa); methylbenzylidene camphor (32a/5pa); octyl methoxycinnamate (3a/4pa); isoamyl p-methoxycinnamate (4a/10pa); and phenylbenzimidazole sulfonic acid (1a/7pa). The frequent (photo)sensitization to isopropyldibenzoylmethane was the reason that its production was discontinued in 1993. 47 patients reacted to fragrance materials, 11 to preservatives and 2 to lanolin alcohol. These constituents were contained in the commercial sunscreens and cosmetics that they had used. Continuous revision of the UV absorber photopatch test series was necessary to be closer to the real frequency of exposure and of reported (photo)allergy to newer sunscreens. Clinicians should consider contact and photocontact allergy, especially in patients with photodermatoses and photo-aggravated dermatoses, and they should perform photopatch testing. Once the culprit has been identified, its INCI (International Nomenclature Cosmetic Ingredients) designation should be given to the patient, who must be warned to avoid products containing the (photo)allergen.

Adolescent↗

PubMed1998

7 years experience of photopatch testing with sunscreen allergens in Sweden.

Since 1990 7 sunscreen allergens have been included in the standard photopatch protocol at 2 Swedish dermatology clinics. 355 consecutive patients with suspected photosensitivity were tested, and in 28 of these (7.9%), a total of 42 allergic reactions were found. 80% of the reactions were of photocontact origin. The most common allergen was benzophenone-3 (Eusolex 4360), with 15 photocontact and 1 contact allergic reactions, followed by isopropyl dibenzoylmethane (Eusolex 8020) (8 photocontact, 4 contact) and butyl methoxydibenzoylmethane (Parsol 1789), with 6 photocontact reactions. There were 2 cases of photocontact allergy to phenylbenzimidazole sulfonic acid (Eusolex 232), which has not been reported previously. 1 case of contact urticaria from benzophenone-3 was accidentally found. In addition, 21 + reactions of doubtful relevance were noted in 14 patients: 16 on irradiated and 5 on non-irradiated test sites. Among these, irritant and phototoxic reactions may be included. These results indicate that the inclusion of UV filters in the standard photopatch protocol is important. Immediate-type testing for urticaria could also be of value.

Adolescent↗

PubMed2006

Causal agents of photoallergic contact dermatitis diagnosed in the national institute of dermatology of Colombia.

OBJECTIVE: To describe and identify the photoallergens causing photoallergic contact dermatitis in the population attending the outpatient clinic of the Centro Dermatologico Federico Lleras Acosta (CDFLLA), the National Institute of Dermatology of Colombia. MATERIALS AND METHODS: Eighty-two patients with clinical diagnosis of photoallergic contact dermatitis enter the study. These patients attended the CDFLLA between August 2001 and May 2003. Photopatch tests were performed using the standard series of sunscreens (Chemotechnique Diagnostics) and 6-methylcoumarin. Cetyl alcohol, phenoxyethanol, methylparabene, propylene glycol, triethanolamine, propylparabene, trichlorocarbanilide and dichromate were also included. The allergens were applied in duplicate on the healthy skin of the back and covered with opaque tape withdrawn 24 h later, the panel on the right was irradiated with an ultraviolet A dose of 5 J/cm(2). The tests were read 24 h after the application of the allergens, 24 and 72 h post-irradiation. The readings were assessed according to the visual scoring system recommended by the International Contact Dermatitis Research Group. RESULTS: Twenty-six patients (31.7%) showed positive photopatch test responses to one or several allergens. Four of them showed positive results to three components of the series and four patients to two components. Thirty-eight photoallergic and 18 allergic reactions were observed. Ultraviolet filters were the substances which more frequently produced positive photopatch test responses (30.5%). The most common ultraviolet filter photoallergen was benzophenone-3 with 22/82 positive results (26.8%), followed by octyl methoxycinnamate (8/82), benzophenone-4 and mexenone (2/82), phenylbenzimidazole sulphonic acid, methylbenziliden camphor and octyl dimethyl PABA (1/82). One patient showed a photoallergic response to 6-methylcoumarin. There was a concordance between the allergen which elicited the positive response and the use of different substances which contained that molecule among its compounds in 17 patients (65.3%). 19.5% of the patients (16/82) showed positive results to one or several allergens in the irradiated panel as well as in the unirradiated control site. These cases were diagnosed as contact allergy, probably caused by aeroallergens, presenting a natural history and a clinical picture similar to photocontact allergy. The most common allergen was dichromate with 10 positive results. CONCLUSIONS: The results of this study confirm that sunscreens are the more frequently involved substances in photoallergic contact dermatitis in our population. Identification of the photoallergen is the key element for adequate disease control and patient education.

Adolescent↗

PubMed1995

Photochemical linkage of antibodies to silicon chips.

Antibodies and antigen binding fragments thereof were photochemically immobilized on surface-modified silicon chips of 5 x 5 mm size. Silicon surface-grafted diazirines and benzophenones formed covalent bonds with the immunoreagents on light activation. Photolithographic immobilization of monoclonal antibodies in aqueous media was achieved on silicon chips by activating surface-grafted benzophenones. The presence of bovine serum albumin during irradiation reduced nonspecific adsorption of the immunoreagents and retained the immunoactivity of the photoimmobilized molecules.

Animals↗

PubMed2002

Identification of the extracellular loop 2 as the point of interaction between the N terminus of the chemokine MIP-1alpha and its CCR1 receptor.

Macrophage inflammatory peptide-1alpha (MIP-1alpha)/CC-chemokine receptor ligand 3 is an 8-kDa peptide that induces chemotaxis of various lymphocytes to sites of inflammation through interaction with the G protein-coupled chemokine receptors CCR1 and CCR5. We recently described the preparation of a photoactivatable derivative of MIP-1alpha labeled with a benzophenone group at the extreme N-terminal end, which is a determinant for the agonist character of chemokines. Benzophenone-MIP-1alpha is a full agonist that specifically and covalently labels CCR1 and CCR5 receptors upon irradiation. In the present study, we use enzymatic and chemical cleavage methods on wild-type and mutated CCR1 receptors to show that the N terminus of the chemokine MIP-1alpha interacts in a specific manner with the second extracellular loop of the CCR1 receptor, within a segment comprising amino acids 178 to 194. This is the first report on the direct identification of a contact point between the N terminus of a chemokine and its membrane-bound receptor. The work shows that the part of chemokines that is endowed with agonist properties interacts with extracellular parts of the receptor rather than the transmembrane core of the protein.

Amino Acid Sequence↗

PubMed1977

Products Formed from Analogues of 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl) Ethane (DDT) Metabolites by Pseudomonas putida.

Cultures of Pseudomonas putida growing in solutions with diphenylmethane as sole carbon source formed 1,1,1',1'-tetraphenyldimethyl ether. The product was identified by gas chromatography, mass spectrometry, and infrared and nuclear magnetic resonance spectrometry. The formation of benzophenone, benzhydrol, and phenylglycolic acid was established by gas chromatography and mass spectrometry. Similar techniques also revealed that phenylacetic acid was a major metabolite. Resting cell suspensions converted benzhydrol to phenyl-glycolic acid and products tentatively identified as hydroxybenzhydrols and a hydroxybenzophenone. Cell suspensions of the bacterium also converted the tetraphenyldimethyl ether to benzhydrol and benzophenone. Possible pathways for the degradation of these analogues of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolites are discussed.

Journal Article↗

PubMedDate not supplied

The stripping technique: in vitro absorption and penetration of five UV filters on excised fresh human skin.

This article gives the results of a study whose aim was to compare the compartmental distribution and absorption of 5 UV filters, in vitro, by fresh human skin, after exposure times of 30 min and 16 h. These UV filters from BASF (octyl methoxycinnamate, benzophenone 4, benzophenone 3, octyl triazone and octocrylene) were incorporated separately in a simple oil-in-water emulsion. The composition of the emulsions was designed in order to obtain a sun protection factor of 5. Therefore the UV filters were introduced into the emulsions at different concentrations. We show that the affinity for each skin level [stratum corneum (SC), viable epidermis, dermis and receptor fluid] is different according to the test substance used. Some substances accumulated in the SC, whereas others passed through the skin very quickly and were quantified in the receptor fluid. The stripping technique allowed us to see that more than 94% of the chemical compound in the SC was in the first eight tapes. The problem of individual values below the limit of detection was raised, a correlation between the two exposure times was found (y = 1.702x - 0.105; R = 0.94) and a classification of products according to their affinity for the SC was determined.

Adult↗

PubMed2000

Absolute rate constants for the quenching of reactive excited states by melanin and related 5,6-dihydroxyindole metabolites: implications for their antioxidant activity.

The triplet-excited state of benzophenone and the singlet-excited state of 2,3-diazabicyclo[2.2.2]oct-2-ene (Fluorazophore-P) have been employed as kinetic probes to obtain information on the antioxidant activity of the skin and eye pigment melanin and its biogenetic precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The excited states were generated by the laser-flash photolysis technique and their reaction kinetics was examined by time-resolved transient absorption or fluorescence spectroscopy, respectively. The reaction between triplet benzophenone and DHI produced with unit efficiency the corresponding 6O-centered semiquinone radical, which was characterized by its characteristic transient absorption. The quenching rate constants for DHI (3.1-8.4 x 10(9) M-1 s-1) and DHICA (3.3-5.5 x 10(9) M-1 s-1) were near the diffusion-controlled limit, indicating excellent antioxidant properties. Kinetic solvent effects were observed. The reactivity of synthetic melanin, assessed through the quenching rate constant of Fluorazophore-P and normalized to the number of monomer units, was more than one order of magnitude lower (2.7 x 10(8) M-1 s-1) than that of its precursors. The trend of the quenching rate constants, i.e. DHI > DHICA approximately alpha-tocopherol > melanin, along with the preferential solubility of DHICA in aqueous environments, serves to account for several experimental results from biochemical studies on the inhibition of lipid peroxidation by these natural antioxidants.

Antioxidants↗

PubMedDate not supplied

Bimolecular hydrogen abstraction from phenols by aromatic ketone triplets.

Absolute rate constants for hydrogen abstraction from 4-methylphenol (para-cresol) by the lowest triplet states of 24 aromatic ketones have been determined in acetonitrile solution at 23 degrees C, and the results combined with previously reported data for roughly a dozen other compounds under identical conditions. The ketones studied include various ring-substituted benzophenones and acetophenones, alpha,alpha,alpha-trifluoroacetophenone and its 4-methoxy analog, 2-benzoylthiophene, 2-acetonaphthone, and various other polycyclic aromatic ketones such as fluorenone, xanthone and thioxanthone, and encompass n,pi*, pi,pi*(CT) and arenoid pi,pi* lowest triplets with (triplet) reduction potentials (E(red)*) varying from about -10 to -38 kcal mol(-1). The 4-methylphenoxyl radical is observed as the product of triplet quenching in almost every case, along with the corresponding hemipinacol radical in most instances. Hammett plots for the acetophenones and benzophenones are quite different, but plots of log k(Q) vs E(red)* reveal a common behavior for most of the compounds studied. The results are consistent with reaction via two mechanisms: a simple electron-transfer mechanism, which applies to the n,pi* triplet ketones and those pi,pi* triplets that possess particularly low reduction potentials, and a coupled electron-/proton-transfer mechanism involving the intermediacy of a hydrogen-bonded exciplex, which applies to the pi,pi* ketone triplets. Ketones with lowest charge-transfer pi,pi* states exhibit rate constants that vary only slightly with triplet reduction potential over the full range investigated; this is due to the compensating effect of substituents on triplet state basicity and reduction potential, which both play a role in quenching by the hydrogen-bonded exciplex mechanism. Ketones with arenoid pi,pi* states exhibit the fall-off in rate constant that is typical of photoinduced electron transfer reactions, but it occurs at a much higher potential than would be normally expected due to the effects of hydrogen-bonding on the rate of electron-transfer within the exciplex.

Calorimetry↗

PubMed2005

Two-generation reproductive toxicity studies in rats with extra parameters for detecting endocrine disrupting activity: introductory overview of results for nine chemicals.

Two-generation reproductive toxicity studies using rats of benzophenone, n-butylbenzene, butyl benzyl phthalate, 2,4-dichlorophenol, dicychlohexyl phthalate, diethyl phthalate, 4-nitrotoluene, lindane and vinclozolin, were performed to investigate whether these chemicals have endocrine-mediated effects with the support of the Ministry of Economy, Trade and Industry and the Ministry of the Environment. Benzophenone exposure was via the diet at concentrations of 0, 100, 450 or 2000 ppm, n-butylbenzene was administered orally by gavage at dose levels of 0, 30, 100 or 300 mg/kg/day, butyl benzyl phthalate was administered orally by gavage at dose levels of 0, 100, 200, or 400 mg/kg/day, 2,4-dichlorophenol was administered in the diet at concentrations of 0, 500, 2000 or 8000 ppm, dicyclohexyl phthalate was given in the diet at concentrations of 0, 240, 1200 or 6000 ppm, diethyl phthalate was administered in the diet at concentrations of 0, 600, 3000 or 15000 ppm, 4-nitrotoluene was administered orally by gavage at doses of 0, 40, 80, or 160 mg/kg/day, lindane exposure was in the diet at concentrations of 0, 10, 60, or 300 ppm, and vinclozolin treatment was by feeding diet at concentrations of 0, 40, 200 or 1000 ppm.

Administration, Oral↗

PubMed1981

Biotransformation of [14C]midazolam in the rat in vitro and in vivo.

1. Aerobic incubation of the maleate salt of midazolam, 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine, with rat liver 9000 g supernatant fluid fortified with an NADPH-generating system yielded three metabolites. These were identified with an 1-hydroxymethyl derivative formed by oxidation of the methyl substituent on the imidazo moiety, the derivative hydroxylated at C-4 of the diazepine ring, and the corresponding dihydroxy derivative. 2. Pretreatment of rats with phenobarbital stimulated the oxidative metabolism in vitro of midazolam by 9000 g liver supernatant. 3. Rats given 2.5 mg/kg of [14C]midazolam maleate i.v. excreted 81% of the administered radioactivity in the faeces and 10% in the urine in 24 h. The highest levels of 14C were in the liver and the lowest in the brain during the first day. 4. Four phenolic derivatives of midazolam were identified in rat bile. These were the 4'-hydroxy and the 4'-hydroxy-1-hydroxymethyl derivatives of midazolam and two benzophenones. Evidence that the benzophenones were artifacts derived from the 4-hydroxy derivatives of the two biliary benzodiazepine metabolites is presented. These biliary metabolites were excreted as glucuronide and/or sulphate conjugates. 5. The Propensity of the rat to excrete phenolic metabolites of midazolam in bile is in agreement with the metabolism of several other benzodiazepines in this species.

Animals↗

PubMed1980

Mass screening and confirmation of diazepam in urine by EMIT-thin-layer chromatography.

A rapid, sensitive, and specific procedure is described for the mass screening and confirmation of diazepam (Valium) in urine at levels as low as 0.5 micrograms/mL. Raw urine specimens are screened directly by the enzyme multiplied immunoassay technique, and those screened positive are confirmed by thin-layer chromatography. Diazepam and its metabolites are converted to benzophenones; diazepam, 3-hydroxydiazepam, and its glucuronide yield 2-methylamino-5-chlorobenzophenone (MACB); whereas N-desmethyldiazepam, oxazepam, and oxazepam glucuronide yield 2-amino-5-chlorobenzophenone (ACB). The two benzophenones are well separated by a solvent system of petroleum ether:glacial acetic acid (90:10) and can be detected under ultraviolet light. A specimen reported positive for diazepam must produce spots for both ACB and MACB. No other benzodiazepines used in the United States or other urinary substances have been found to interfere; thus the procedure described is specific for for diazepam.

Chromatography, Thin Layer↗