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Computer-aided identifications of thin-layer chromatographic patterns in broad-spectrum drug screening.

Numerous analytical schemes have been designed to test for drugs in the general unknown context by thin-layer chromatography (TLC TLC total lung capacity; thin-layer chromatography.

TLC
abbr.
1. thin-layer chromatography

2.
) [1-4], gas-liquid chromatography [5], HPLC HPLC high-performance liquid chromatography.

HPLC

high performance liquid chromatography.

HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed
 [6], GC/MS GC/MS Gas Chromatograph/Mass Spectrometer
GC/MS Gas Chromatograph/Mass Spectrometry
GC/MS Gas Chromatograph/Mass Spectrograph
 [7], and combinations thereof. (1) Carvalho et al. [8] carried out qualitative TLC with color reactions and used a computer program to sort data for 37 drugs. However, their technique required four solvent systems and was limited to 37 drugs. ANSYS ANSYS Analysis System , Inc., introduced Toxi-Lab in 1981 [9] as a commercially available technique for systematic drug detection and identification and provided a compendium illustrating color reactions of drugs. Toxi-Lab is efficient but has marginal sensitivity for many drugs [2, 3, 9,10], poor resolution when several drugs are present, and depends on one color reaction (which gives no color with some drugs) and one general staining technique that reacts with nearly all drugs. The accompanying computer program (TOXI-SEARCH) is not currently advertised by ANSYS.

Our goals with the technique presented here included having as many unique sets of reactions as possible (high specificity) within 1-2 h of one chemist's bench time and using routine silica-gel TLC plates and other materials readily available in most toxicology laboratories. We also wanted to meet the criterion that general unknown screening should logically determine what is not present as well as what is present through the use of reliable exclusion and accurate positive identifications of xenobiotics. The training in TLC required to make accurate identifications has been an important factor in developing the technique we present. A computer program, SPOT CHEK, was developed to minimize the time spent interpreting data by tediously matching observed TLC migration and reaction characteristics with those previously obtained for drug standards. Detection reagents were chosen to detect 100 ng or less per applied spot of sample for a wide variety of drug groups. Results for 9 TLC reactions and the migration position (the 10th defining variable) relative to nicotine for each drug or drug metabolite have been placed in the database to facilitate a search keyed on any one (or more) of these 10 TLC identifying characteristics. After the search is completed, the program presents a list of the matches between test specimen substances and database entries.

Materials and Methods

TLC

Supplies and equipment. All separations and reactions were carried out on scored hard-layer silica-gel plates (prod. no. 47521; Analtech, Newark, DE). The plates were developed in two-sided chambers with stainless-steel covers (no. 022.5155; CAMAG Scientific, Wilmington, NC). The double-sided TLC chamber allows for a 10-min solvent-preloading time for the plate before development is started. Spray bottles, paper saturation pads, dipping containers, a UV viewing chamber (Spectroline CM-10), and a hand-held UV 254/366-nm lamp (Spectroline ENF ENF Enforcement
ENF Enforcement Action (Environmental Term AR 200-1)
ENF Elks National Foundation
ENF Éclaireurs Neutres de France (French Scouting association)
ENF Event Notification Facility
 240-C) were supplied by Alltech (State College, PA). Other glassware included pipets, capillary pipets for sample application, conical plastic disposable evaporation cups (Har-Len Medical, Pittsburgh, PA), and screw-cap jars containing various reagents into which the TLC plates would be dipped.

Detection reagents. The 10 TLC detection/visualization reagents used (SPOT ID reagents) were prepared with minor modification of the formulations given elsewhere [111. Composition of the reagents was as follows:

S1, fluorescamine (Sigma Chemical Co., St. Louis, MO), 0.3 g/L; stable 1 month stored in a brown bottle

S2, ferric chloride /perchloric acid/nitric acid (FPN FPN Fixed Pattern Noise (variation of the pixel response at one moment of an image sensor in the dark)
FPN Fixed Penalty Notice (UK law enforcement)
FPN Fundación Patagonia Natural
FPN Fine Print Note
) reagent: 90 mL of 70% perchloric acid, 135 mL of 70% nitric acid, and 15 mL of 50 g/L ferric chloride added to 100 mL of water; stable 6 months in a brown bottle

S3, Dragendorff reagent, prepared as in Clarke [11]

S4, Marquis reagent: concentrated sulfuric acid

S5, Mandelin reagent: ammonium metavanadate (A8050; Sigma Chemical Co.), 800 mg/L in concentrated sulfuric acid; stable at room temperature for at least a year

S6, iodinated Dragendorff reagent: 5 g of potassium iodide, 2 g of iodine, and 0.2 g of bismuth subnitrate (Sigma B9009) mixed in water, followed by 0.5 mL each of glacial acetic acid glacial acetic acid
n.
Acetic acid that is at least 99.8 percent pure.
 and concentrated HCI (Human Computer Interaction) Refers to the design and implementation of computer systems that people interact with. It includes desktop systems as well as embedded systems in all kinds of devices. , and brought to 250 mL with deionized water

S7, 254 nm (UV) light projected onto a developed TLC plate (must be fluorescent indicator plate)

S8, chlorine vapor, produced as needed by adding concentrated HCl to commercial bleach (5% hypochlorite hypochlorite /hy·po·chlo·rite/ (-klor´it) any salt of hypochlorous acid; used as a medicinal agent with disinfectant action, particularly as a diluted solution of sodium hypochlorite. )

S9, concentrated HCI fumes, produced as needed in a fume in ill temper, esp. from impatience.

See also: Fume
 hood

S10, the mobile phase (described below). Names, application technique, and shelf-life of the reagents are given in Table 1. Before dipping plates 2B and 2C into S4 and S5 reagents, we exposed them for ~5 min to vapors from formaldehyde /methanol solution (37/63 by vol.), then dipped them into the respective acid solutions. Mercuric mercuric /mer·cur·ic/ (mer-kur´ik) pertaining to mercury as a bivalent element.

mer·cu·ric
adj.
Relating to or containing mercury, especially with a valence of 2.
 sulfate sulfate, chemical compound containing the sulfate (SO4) radical. Sulfates are salts or esters of sulfuric acid, H2SO4, formed by replacing one or both of the hydrogens with a metal (e.g., sodium) or a radical (e.g., ammonium or ethyl).  (not one of the 10 SPOT ID reagents) was prepared by dissolving 5 g of mercuric sulfate in water plus 20 mL of concentrated sulfuric acid and bringing to 250 mL with water (stable at least a year).

For all chromatographic chro·mat·o·graph  
n.
An instrument that produces a chromatogram.

tr.v. chro·mat·o·graphed, chro·mat·o·graph·ing, chro·mat·o·graphs
To separate and analyze by chromatography.
 runs we used a single mobile phase: ethyl acetate/ dichloromethane/methanol/concd. ammonium hydroxide (80/90/15/5 by vol.). Migration of drug substances was defined by a "zone," or position relative to a set of known substances present in the normalizing (N) mixture. The compounds included in the N mixture were (Fig. 1, reading spots from bottom to top): benzoylecgonine, morphine, quinine quinine (kwī`nīn', kwĭnēn`), white crystalline alkaloid with a bitter taste. Before the development of more effective synthetic drugs such as quinacrine, chloroquine, and primaquine, quinine was the specific agent in the treatment of , nicotine, methadone methadone (mĕth`ədōn', –dŏn'), synthetic narcotic similar in effect to morphine. Synthesized in Germany, it came into clinical use after World War II. It is sometimes used as an analgesic and to suppress the cough reflex. , and cocaine. The area from the origin to the center of the morphine spot was designated Zone 1, from morphine to quinine Zone 2, from quinine to nicotine Zone 3, from nicotine to cocaine Zone 4, and from cocaine to the solvent front Zone 5. Substances that eluted even with a boundary line were referenced to the higher-numbered zone. The TLC chamber must be two-sided or have a small container in which to place mobile phase for preassay vapor-loading of the plate. The calibrating mixtures in M and P (Fig. 1) are neutral drugs (M mixture) and barbiturates (P mixture); [S.sub.1], [S.sub.2], and [S.sub.3] are benzodiazepines and other neutral/acidic drugs; A, B, and C mixtures are basic amine-type drugs. The total number of individual drugs present in the set of mixtures is 48, each at 1 g/L in methanol. Mixture compositions are given below under Assay Performance (Separations) and in Fig. 1.

COMPUTER AND COMPUTER PROGRAM

Data for >240 drug substances have been placed into a PC program, compressed, compiled, and installed onto an MS DOS hard drive, version 6.2. The original program, SPOT CHEK, was written in dBase IV and requires an IBM (International Business Machines Corporation, Armonk, NY, www.ibm.com) The world's largest computer company. IBM's product lines include the S/390 mainframes (zSeries), AS/400 midrange business systems (iSeries), RS/6000 workstations and servers (pSeries), Intel-based servers (xSeries)  or IBM-compatible 286 or higher PC, DOS 6.0 or later 100%-compatible version, at least 640 K of memory plus 2 Mb of extended RAM, at least 3 mB of hard disk space (4.5 mB at installation), and an CGA, EGA, or VGA (Video Graphics Array) The display standard for the PC. All PC display adapters support VGA, and Windows machines boot up in "VGA mode" before switching to higher resolutions.  monochrome or color monitor. A peripheral printer is optional but is useful for printing lists of matches after data entry. Data cards. TLC reactions were detailed on 250 separate identification cards that give all of the information database for each drug. Fig. 2 shows as an example the data card for carbamazepine carbamazepine /car·ba·maz·e·pine/ (kahr?bah-maz´e-pen) an anticonvulsant and analgesic used in the treatment of pain associated with trigeminal neuralgia and in epilepsy manifested by certain types of seizures. . All substances have a relative [R.sub.f], nicotine being assigned [R.sub.f] = 1.0. The data cards are indexed alphabetically and sorted in order of increasing relative [R.sub.f]. As shown in Fig. 2, the data cards contain, in addition to the 10 reactions in SPOT CHEK, information that will greatly assist in making accurate drug identifications. Data cards illustrate portions and reactions of metabolites of parent drugs, list drugs that react similarly (can be mistaken for suspect drug), give the most sensitive reagents for visualization, and offer important details of the TLC behavior of the various drugs.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

SAMPLE PREPARATION/ DRUG RECOVERY FROM URINE AND SERUM

An aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share)  of serum (0.5-1 mL) not pH-adjusted was extracted with 6 volumes of a solution of dichloromethane/isopropanol (19/1 by vol.). Whole blood was diluted with 1 volume of water before extraction with the 6 volumes of solvent. Two 5-mL aliquots of urine, one adjusted to pH 9 and the other to pH 12, were extracted with 10 mL of the dichloromethane/isopropanol solvent and combined into one extract fraction. The serum and urine extracts were passed through coarse filter paper and evaporated to dryness in disposable plastic 16-mL conical beakers. Before evaporation, two drops of 1 mL/L HCl in methanol were added to urine extracts to prevent loss of volatile amines. This isolation technique provides a weak acid/neutral fraction and an amphoteric/amine base fraction. The serum extract, which might contain neutral drugs, weakly acidic drugs (barbiturates, acetaminophen acetaminophen (əsēt'əmĭn`əfĭn), an analgesic and fever-reducing medicine similar in effect to aspirin. It is an active ingredient in many over-the-counter medicines, including Tylenol and Midol. ), and benzodiazepines, was spotted onto Plates 1A and 1B (Fig. 1), and the basic /amphoteric fraction from urine was spotted onto Plates 2A, 2B, and 2C.

We examined some drugs--from very polar (benzoylecgonine) to nonpolar nonpolar

not having poles; not exhibiting dipole characteristics.
 (phencyclidine phencyclidine /phen·cy·cli·dine/ (PCP) (fen-si´kli-den) a potent veterinary analgesic and anesthetic, used as a drug of abuse in the form of the hydrochloride salt; its abuse by humans may lead to serious psychological disturbances. )--for approximate recoveries in the liquid-liquid extraction technique described above. Because on the silica-gel TLC plate the drugs migrate according to their polarity, recovery correlates well with migration position; i.e., recovery increases as [R.sub.f] increases. Benzoylecgonine, found low in Zone 1, has ~45% recovery and phencyclidine ~95% recovery. Morphine recovery is ~70%, and drugs migrating farther than morphine are 75-95% recovered. Benzodiazepines, barbiturates, acetaminophen, caffeine, and other neutral compounds and weak acids are recovered from serum in high yield (>80%) with no pH adjustment. Recovery was determined by measuring the fraction remaining unextracted after the initial extraction.

ASSAY AND TECHNIQUES

Application of samples. The plates used throughout were factory-scored hard-layer silica-gel plates, 20 cm wide X 10 cm high (running distance), with an organic binder and fluorescent indicator. Biological fluid extracts, powders, and other solid dosage forms (e.g., tablets, pills, residues) were applied directly to the plates in methanol or dichloromethane. Residues from both the neutral and the pH 9 plus 12 fractions were taken up in a few drops of dichloromethane/isopropanol solvent and applied in 10-[micro]L capillaries or by an automated applicator ap·pli·ca·tor
n.
An instrument for applying something, such as a medication.


applicator,
n a device for applying medication; usually a slender rod of glass or wood, used with a pledget of cotton on the end.
. The neutral extract was spotted about equally on Plates 1A and 1B and the pH 9 plus 12 fraction from urine was spotted about equally on Plates 2A, 2B, and 2C, ~20% of this residue being reserved for optional tests. The plates being spotted were placed over a 2.54-cm-wide heating tape so that application spots dried quickly and fit within a standard 3-mm-diameter size for mixtures and extraction fractions (see Fig. 1 for spotting pattern). The scored plates were sectioned after development, except that Plate 2C (Fig. 1) was separated from the other sections before development. This permitted the placing of all 5 sections of plate into a single small development chamber (2C was placed facing the other plate sections) and developed in the mobile phase described above. Serum and urine extracts from one or two subjects were accommodated on a 12.5 X 10 cm (w x l) plate. Larger plate sections of 1A, 1B, 2A (etc.) and tanks holding 20 X 10 cm plates were used when we were analyzing specimen extracts from more than two subjects.

Plate development. A paper pad was placed on one side of a two-sided TLC chamber for 10 x 10 cm plates, and 6 mL of developing solvent was run (pipetted) down the pad, followed by 3 drops of concentrated N[H.sub.4]OH (pad side). The plate was placed on the dry side of the chamber for 10 min before developing to full plate height by the addition of 6 to 8 mL of solvent. Chromatography per se took 10.5 min. The plates were then dried and observed in the viewing chamber at 254 and 366 nm, where we marked the UV-quenching spots lightly with a pencil and made note of any fluorescent spots. Relative [R.sub.f] values were not routinely measured, but zone positions were accurately noted by reading the positions of detected substances vs those of the N calibrator calibrator

an instrument for dilating a tubular structure or for determining the caliber of such a structure.
 drugs, either under the UV 254 light (reaction S7) or after a visualization reaction.

Plate reactions. Reagents were applied (after segmenting the plates as in Fig. 1) by classical techniques, either dipping or spraying as indicated in Table 1. Barbiturates, meprobamate meprobamate (məprō`bəmāt'), tranquilizing drug that acts as a depressant of the central nervous system and is commonly used in the treatment of anxiety and sometimes schizophrenia. , glutethimide, and other nonbarbiturate sedatives were detected or ruled out by use of mercuric sulfate dip (neutral/weak acid extract) on plate 1A. After positioning plate 1B over a 600-mL beaker on a warm hot plate (in a fume hood), we added several milliliters of commercial bleach followed immediately by the same volume of concentrated HCl. This produced sufficient [Cl.sub.2] gas to react with drugs on the developed plate. After exposure to [Cl.sub.2] vapor for 10-15 s, some drugs give a visible color (acetaminophen), whereas others fluoresce fluo·resce  
intr.v. fluo·resced, fluo·resc·ing, fluo·resc·es
To undergo, produce, or show fluorescence.



[Back-formation from fluorescence.
 strongly (some benzodiazepines, carbamazepine). We next exposed plate 1B to HCl vapors (in a fume hood). After each reaction ([Cl.sub.2] and HCl) we examined plate 1B for fluorescence with UV 254 and 366 nm light. Detection is vastly improved by placing the plate over a hand-held UV lamp (face up) inside the dark chamber rather than having the light source above the plate.

Plates 2A, 2B, and 2C were processed according to the scheme in Fig. 1 and all reactions were noted. The sequence of reactions for plate 2 was as follows: 2A, N[H.sub.4]OH vapor, fluorescamine, FPN, D1; 2B, formaldehyde for 5-10 min, then concentrated [H.sub.2]S[O.sub.4] dip; 2C, formaldehyde for 5-10 min, then Mandelin reagent dip. After the acid dips for 20-50 s, plates 2B and 2C were dipped first into water and then into reagent D2 for 2-5 min, which fixes the location of most drug substances. The two acid dips produce colors in the spots, so a color number is assigned to all spots on the plates that give colors with the acid dips. The color code was: 1, pink; 2, red; 3, copper/rust/red-brown; 4, orange/peach/yellow-orange; 5, tan/salmon/gold; 6, yellow; 7, green; 8, olive green/gray-green; 9, blue-green/aqua/lime; 10, blue (light and dark); 11, violet/purple/lavender; and 12, gray/blue-gray. Reaction results and the zone positions of unknown spots on the plate were recorded. These nine reactions plus the zone position, collectively called SPOT ID reactions, are numbered S1 through S10 for convenient tabulating and entry into the SPOT CHEK program.

ASSAY PERFORMANCE

Separations. The silica-gel plates and mobile phase used resolved individual drugs within classes sufficiently so that 48 drugs seen very frequently could be applied as calibrators and fully separated. Mixtures of these calibrators [1 g/L (1 [micro]g/[micro]L) each] were applied to TLC plates as long as 2 weeks before plate development, ~2 [micro]g per spot. No decomposition of the calibrators occurred if the prepared plate was covered with a glass plate and kept in a closed box. The components of each calibration mixture are listed in the legend to Fig. 1. This large number of calibrators allows for visual determination that an unknown drug is between two calibrators rather than measuring the [R.sub.f] or relative [R.sub.f] of the unknown. For example, for a drug that migrates between methadone and cocaine (in the N mixture), this area of the TLC plate presents few matches. Migration relative to a known calibrator is more consistent than relative [R.sub.f], which varies as much as 15% from plate to plate. Some drugs are more susceptible to slight solvent changes than others. For example, phenobarbital is moderately dependent on the ammonia concentration within the chamber, and benzoylecgonine migration depends on the moisture content of the TLC plate. Whereas relative [R.sub.f] numbers can change from plate to plate, migration positions relative to the N mixture set remain constant if the developing conditions are according to the protocol given above.

Detection limits. Detection limits, in terms of quantity (ng)/spot, are dependent on spotting technique and the reaction used to visualize the drugs. Table 2 indicates the approximate detection limits expected for several drugs and visualization reagents, assuming that the procedure is performed by someone with reasonable analytical skill and experience. Detection limits in terms of concentrations in urine and serum (ng/mL specimen) are dependent on recovery, spotting technique, and reagent for visualization. Tables 3 and 4 present results for several drugs added to 0.5-mL serum and 5-mL urine samples and are typical of results for all 240 drugs studied. These results should be considered approximate because experience, plate quality, and TLC skills are factors in the overall detection. Nevertheless, the technique presented here offers lower detection limits for all classes of drugs relative to previously published detection limits [2, 3, 9,10,121, either per spot or per volume of specimen.

Validation. Data on many drugs and drug metabolites have been accumulated over several years in the process of broad-based drug screening of >15 000 individual urine and serum/blood sets. Validation of the TLC techniques was accomplished by using at least one of several alternative techniques-RIA, gas chromatography, HPLC, UV spectrophotometry spectrophotometry

Branch of spectroscopy dealing with measurement of radiant energy transmitted or reflected by a body as a function of wavelength. The measurement is usually compared to that transmitted or reflected by a system that serves as a standard.
, fluorescence polarization immunoassay Immunoassay

An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus.
 (FPIA FPIA Family Planning International Assistance
FPIA Fort Payne Improvement Authority
FPIA Flourescence Polarization Immunoassay
), and GC/MS--to confirm the TLC identifications. All positive results for cocaine /benzoylecgonine, opiate opiate /opi·ate/ (o´pe-it)
1. any drug derived from opium.

2. hypnotic (2).


o·pi·ate
n.
1.
, barbiturate barbiturate (bärbĭch`ərāt'), any one of a group of drugs that act as depressants on the central nervous system. High doses depress both nerve and muscle activity and inhibit oxygen consumption in the tissues. , phencyclidine, amphetamine amphetamine (ămfĕt`əmēn), any one of a group of drugs that are powerful central nervous system stimulants. Amphetamines have stimulating effects opposite to the effects of depressants such as alcohol, narcotics, and barbiturates. , and benzodiazepine benzodiazepine (bĕn'zōdīăz`əpēn'), any of a class of drugs prescribed for their tranquilizing, antianxiety, sedative, and muscle-relaxing effects. Benzodiazepines are also prescribed for epilepsy and alcohol withdrawal.  were tested by RIA; tricyclics, barbiturates, and various neutral drugs positive by TLC were tested by FPIA; drugs with unique UV spectra such as naproxen naproxen and naproxen sodium, potent nonsteroidal anti-inflammatory drugs (NSAID) used to alleviate the minor pain of arthritis, menstruation, headaches, and the like, and to reduce fever.  and methaqualone meth·a·qua·lone
n.
A potentially habit-forming drug used as a sedative and hypnotic.



methaqualone

a nonbarbiturate hypnotic similar to barbiturates in its effects.
 were scanned by UV spectrophotometry; gas chromatography, GC/MS, or HPLC was used in numerous instances where TLC findings were not clearcut.

The Toxi-Lab compendium, consisting of color photographs of spots after the Mandelin reaction, helped us confirm how drugs and metabolites react with this reagent. Urines from persons taking a single known medication were processed on several occasions to obtain metabolite patterns (e.g., for tramadol, ibuprofen ibuprofen (ī`byprō'fən), nonsteroidal anti-inflammatory drug (NSAID) that reduces pain, fever, and inflammation. , and codeine codeine (kō`dēn), alkaloid found in opium. It is a narcotic whose effects, though less potent, resemble those of morphine. An effective cough suppressant, it is mainly used in cough medicines. Like other narcotics, codeine is addictive. ). Patient histories in which drug intake was reasonably documented provided a check on the overall reliability of the SPOT CHEK technique. Information for the data cards (Fig. 2) was based on positive, confirmed identifications with authentic drugs.

Specificity. Identification problems most often involved choices between closely related drugs or drugs with few or no SPOT ID reactions other than migration position. For example, orphenadrine and diphenhydramine diphenhydramine /di·phen·hy·dra·mine/ (di?fen-hi´drah-men) a potent antihistamine, used as the hydrochloride salt in the treatment of allergic symptoms and for its anticholinergic, antitussive, antiemetic, antivertigo, and antidyskinetic  have very similar reagent reactions but can be differentiated by slight migration differences. Brompheniramine, pheniramine, and chlorpheniramine are difficult to distinguish by these TLC techniques and when present in a mixture could not be resolved with the solvent system presented here.

Persons who are color-blind can use SPOT CHEK, but they need to use the Marquis and Mandelin color codes guardedly or have others' eyes note the colors.

DATA ENTRY AND EFFECTIVE USE OF SPOT CHEK COMPUTER PROGRAM

To operate the program, the user notes systematically the reactions and zone for each spot. Weak reactions are noted as such and colors are described in words, then assigned the corresponding number from the color key. The program, installed in MS DOS onto a PC, is then opened. The result of each SPOT ID reaction (the 9 reactions) is asked for with a yes/no (Y/N Y/N Yes/No ) statement or a prompted color number (reactions 4 and 5), as follows.

S1, fluorescamine reaction-Quench, Fluoresce, None? (Q/F/N)

S2, FPN reaction? (Y/N)

S3, D1 Dragendorff reaction ? (Y/N)

S4, Marquis reaction? Enter number corresponding to color, or NN for no color

S5, Mandelin reaction? Enter number corresponding to color, or NN for no color

S6, D2-iodinated Dragendorff reaction? (Y/N)

S7, UV 254 nm reaction-quench fluorescence? (Y/N)

S8, chlorine vapor reaction? (Y/N)

S9, HCl vapor reaction? (Y/N)

S10, zone portion on plate? Enter 1, 2, 3, 4, or 5

Would you like a printout? (Y/N)

Are all entries correct ? (Y/N)

If N (no), go back to start (S1). Initially, only those reactions that were carried out and gave very clearcut spots should be entered. Leaving the response blank means that the reaction will not be considered. After the last entry, the program produces on the screen a list of drugs matching the designated properties. Only if the list contains more than four entries is one advised to enter more reactions. Specificities and probability matches vary with the compound and to some extent with the skill and experience of the analyst. Once a small list has been generated, the data cards are to be examined carefully for best matches and for those substances similar to the suspect drug.

Results

Table 5 gives the results from seven individual drug screens processed according to the procedure given and to the TLC reactions entered into the SPOT CHEK computer program. Not all reactions were used for the individual cases; in some cases, only a single plate was used to acquire a listing or identification. After obtaining a list, whether one drug or more, we compared each drug with a data card of the listed drugs such as the one given in Fig. 2. The exact position ([R.sub.f] relative to nicotine) and the metabolite pattern assisted in distinguishing close matches. Case 1 was from a pH 7 serum extract spotted onto plates 1 and 2 (Fig. 1). If only reactions S6, S7, S8, S9, and Zone are entered into SPOT CHEK, the search list is too long for quick identification. By including plates 2A and 2B, the program lists four possible drugs, which are readily differentiated by close inspection of the TLC plate for relative [R.sub.f] values of the three benzodiazepines and carbamazepine, all having been present in the calibrators applied. In case 2, SPOT CHEK selected dextromethorphan as the only choice after plates 2A, 2B, and 2C were developed, reacted, and read. The acid-neutral side of the reaction system (plate 1A and 1B) was not needed to select dextromethorphan: Basic drugs can usually be identified with plates 2A, 2B, and 2C, whereas benzodiazepines and various neutral compounds can be detected and identified on Plate 1. The third set of plates (case 3) initially used plate 2A, which resulted in 12 possible identifications. Adding plate 2B shortened the selection list to 2 substances but did not reduce to 1 "hit." Guaifenesin and methocarbamol can be differentiated with p-dimethylaminobenzaldehyde, because methocarbamol is a carbamate carbamate /car·ba·mate/ (kahr´bah-mat) any ester of carbamic acid.

car·ba·mate
n.
A salt or ester of carbamic acid.
 and guaifenesin is not.

Some drugs can be selected on the basis of only one TLC plate, e.g., case 4 in Table 5. The absence of UV absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .

2.
 and the charring with FPN reagent are highly characteristic of erythromycin erythromycin (ĭrĭth'rōmī`sĭn), any of several related antibiotic drugs produced by bacteria of the genus Streptomyces (see antibiotic). . The last three searches in Table 5 resulted in single-drug listings: case 5, azacyclonol; case 6, lidocaine lidocaine /li·do·caine/ (li´do-kan) an anesthetic with sedative, analgesic, and cardiac depressant properties, applied topically in the form of the base or hydrochloride salt as a local anesthetic; also used in the latter form as a  metabolite (monoethylglycinexylidine or MEG-X), and case 7, amiodarone. Not all reactions were required for the specific identification of these basic drugs.

Discussion

By accumulating TLC information systematically in a PC database, we have developed a convenient program, meaningful to chemists, for assisting in the detection and identification of large numbers of drugs, as would be sought in general unknown screening. The approach can be applied to urine extracts, serum/blood extracts, solid dosage forms (e.g., tablets, pills), and mystery powders. The logic of the SPOT CHEK technique is towards discovery of drug classes (e.g., benzodiazepines, barbiturates), functional groups (primary, secondary, and tertiary amines), and conjugation conjugation, in genetics
conjugation, in genetics: see recombination.
conjugation, in grammar
conjugation: see inflection.
 (as in aromatic structures) by fluorescamine, FPN, and Dragendorff reagents and UV absorbance. Color reactions lend high specificity to the technique. Speed (bench time) and sensitivity (detection of low nanogram nanogram /nano·gram/ (ng) (nan?o-gram) one billionth (10-9) of a gram.

nan·o·gram
n. Abbr. ng
One billionth (10-9) of a gram.
 quantities) are not sacrificed by the TLC technique described. Moreover, new drugs can be readily added to the database: Most drugs will react in some way with one of the reagents used. Drug identifications in simple one- or two-drug urine specimens, such as those obtained in proficiency tests, are readily accomplished by the TLC technique presented here. However, experience with the technique is required when several drugs-and their metabolites-are present in a urine extract. SPOT CHEK can be used for selective drug screens by using the most sensitive or selective reagents for the drugs that are to be included in screening.

The technique described here can be carried out without the assistance of the computer program. Advantages of the TLC technique alone are: (a) in a single developing system, plates provide good separations for drug substances and have a high peak (spot) capacity; (b) plates are convenient to use because ruggedness of the layer allows dips and sprays to be applied without disintegrating the layer; (c) the last reaction of each sequence can be read weeks after it has been carried out; (d) reagents used will detect drugs in quantities <100 ng/spot, in some cases as little as 5 ng/spot; (e) scored plates are efficient and economical; (fl reagents are stable and do not need to be made up daily, (g) new drugs can be readily added to the total screen, and (h) the drug recovery technique is flexible; i.e., laboratories can continue in-house procedures or use the one described here. However, the tedious and somewhat subjective interpretation of TLC results can now be made faster and more objective by utilization of a versatile but simple computer program SPOT CHEK.

In the two papers published on the use of a computer for storage and retrieval of TLC data [8,13], both studies rely on [R.sub.f] determinations in several mobile-phase solvents, thereby increasing analysis time. The SPOT CHEK program, however, has the following advantages: (a) from 1 to 10 TLC characteristics (identification variables) can be entered, depending on how many reactions were done (i.e., partial use of the system is feasible); (b) the program works in practical situations, as demonstrated by several years of experience with >15 000 full drug screens from clinical and forensic referrals; (c) the program will often list results by drug class; (d) the identification technique is reaction-based and visual and does not require tedious measurements of migration positions or the use of more than one mobile phase. The program is also very helpful in training analysts in TLC and in understanding drug chemistry. Finally, and most importantly, the computer-assisted interpretation of results obviates much of the tedium involved in broad-spectrum drug screening.

In conclusion, the extent and degree of confirmation of drug substances identified or selected by the TLC technique described here depend on how the results are to be used. The decision of how and when to confirm drug finding will depend on laboratory protocols and on the chemist's perspective, experience, and insight. Some of the drugs in the SPOT CHEK database and card file are uniquely defined by their TLC reactions and metabolite patterns. The approach of Ojanpera and Vuori [4], who presented TLC identifications by scanning individual spots in situ in the UV range, is especially compatible with the SPOT ID reactions and SPOT CHEK program presented here because the UV spectrum can be obtained nondestructively. With a "hit" list from a UV scan, selective SPOT ID reactions can follow. TLC offers an excellent first step in broad-spectrum drug screens and in general unknown protocols; once applied to the plate, the sample is not lost (unless volatile). The techniques to corroborate To support or enhance the believability of a fact or assertion by the presentation of additional information that confirms the truthfulness of the item.

The testimony of a witness is corroborated if subsequent evidence, such as a coroner's report or the testimony of other
 TLC findings can be intelligently selected because (after TLC) the analyst will know something about the functional groups, aromaticity, polarity, and often the likely identification of the unknown(s) before the confirmatory step. Non-TLC confirmatory techniques appropriate in clinical and forensic toxicology include immunoassay (RIA, FPIA, Emit), UV spectrophotometry, gas and liquid chromatography, and GC /MS.

We dedicate this paper to the memory of E. Paul Elson, the PC specialist and author of the original SPOT CHEK software program written in dBase IV. Revisions of the program were made by us.

References

[1.] Moffat AC. The standardization of chromatographic systems for the identification of basic drugs. J Chromatogr 1975;110:341-7.

[2.] Jones DW, Adams D, Martel PA, Rousseau RJ. Drug population in 1000 geographically distributed urine specimens. J Anal Toxicol 1985;9:125-30.

[3.] Singh AK, Granley K, Ashraf M, Mishra U. Drug screening and confirmation by thin layer chromatography Thin Layer Chromatography (TLC) is a chromatography technique used to separate chemical compounds [1]. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose immobilised onto a flat, . J Planar Chromatogr 1989;2:410-9.

[4.] Ojanpera I, Vuori E. Identification of drugs in autopsy liver samples by instrumental qualitative thin-layer chromatography. J Chromatogr 1994;674:147-52.

[5.] Drummer OH, Horomidis S, Kourtis S, Slyrjanen ML, Tippett P. Capillary gas chromatographic drug screen for use in forensic science. J Anal Toxicol 1994;18:134-8.

[6.] Drummer OH, Kotsos A, McIntyre M. A class-dependent drug screen using a photodiode A light sensor (photodetector) that allows current to flow in one direction from one side to the other when it absorbs photons (light). The more light, the more the current. Used to detect light pulses in optical fibers and other light-sensitive applications, it works the opposite of a  array detector. J Anal Toxicol 1993;17: 225-9.

[7.] Smith NB. Automated identification by computer of the mass spectra of drugs in urine of serum extracts. J Anal Toxicol 1994;18:16-21.

[8.] Carvalho TMJP, de Alvencar JW, Machado MIL, Coelho HLL HLL - high-level language . Drug analysis by thin-layer chromatography and with the aid of a microcomputer. Rev Bras Farm 1994;75(1):25-8.

[9.] Bonicamp JM, King DL, O'Brien BA, Jones DW. A simple procedure for separating drugs from interfering lipids on special thin layer chromatographic media. J Anal Toxicol 1981;5:262-3.

[10.] O'Brien BA, Bonicamp JM, Jones DW. Differentiation of amphetamine and its major hallucinogenic hal·lu·ci·no·gen  
n.
A substance that induces hallucination.



[hallucin(ation) + -gen.]


hal·lu
 derivatives using thin-layer chromatography. J Anal Toxicol 1982;6:143-6.

[11.] Moffat AC, ed. Clarke's isolation and identification of drugs, part 4, 2nd ed. London: Pharmaceutical Press, 1986:1169-71.

[12.] McElwee DJ. A new method for the detection of sedative sedative, any of a variety of drugs that relieve anxiety. Most sedatives act as mild depressants of the nervous system, lessening general nervous activity or reducing the irritability or activity of a specific organ.  drugs on thin-layer chromatograms using chlorine vapors and 2,7-dichlorofluorescein. J Anal Toxicol 1979;3:266-8.

[13.] Gill R, Brown C, Moffat AC. A computer search system for the identification of drugs using a combination of thin-layer chromatographic gas-liquid chromatographic and ultraviolet spectroscopic spec·tro·scope  
n.
An instrument for producing and observing spectra.



spectro·scop
 data. Analyst 1985;110:1059-65.

THEODORE J. SIEK,* COREY W. STRADLING, MARC W. MCCAIN, and TIMOTHY C. MEHARY

Analytic Bio-Chemistries, Inc., 1680-D Loretta Ave., Feasterville, PA 19053.

* Author for correspondence. Fax 215-322-4226.

(1) Nonstandard non·stan·dard  
adj.
1. Varying from or not adhering to the standard: nonstandard lengths of board.

2.
 abbreviations: TLC, thin-layer chromatography; FPN, ferric ferric (fĕr`ĭk), iron in the +3 valence state.


See ferrous.
 chloride/perchloric acid/nitric acid; FPIA, fluorescence polarization immunoassay.

Received June 11, 1996; revised November 4, 1996; accepted November 21, 1996.
Table 1. TLC reagents used to detect chromatographed
drug substances and application-visualization technique.

    Reagent       SPOT ID code        How applied        Shelf life

Fluorescamine     S1              Dip or spray           1 month
                  fluorescamine
FPN               S2 FPN          Light spray, heat      6 months
Dragendorff       S3 D1           Dip or spray           1 year
Marquis (a)       S4 & color      Dip only               1 month
Mandelin (a)      S5 & color      Dip only               1 month
Dragendorff +
  [l.sub.2]       S6 D2           Dip or spray           1 year
UV 254 nm light   S7 UV 254 nm    Dark viewing box       --
Chlorine vapor    S8 [Cl.sub.2]   [Cl.sub.2] as needed   b
HCI vapor         S9 HCI          HCI as needed          b
Migration zone    S10 Zone        Migration position     b
Mercuric
  sulfate (d)     Extra reagent   Dip or spray           1 year

(a) The developed plate was exposed to formaldehyde vapor
in a screw-cap bottle for 3-10 min and then was dipped into
concentrated [H.sub.2][SO.sub.4] (S4, Marquis reaction); a
second plate similarly exposed to formaldehyde was dipped
into Mandelin reagent (S5). Specific colors are noted with
the S4 and S5 reactions and compared with the color code
stated in the text.

(b) Vapors of [Cl.sub.2] and HCl are produced in a fume hood
as described under Assay and Techniques, Plate reactions.

(c) Migration zone ranges from 1 to 5, according to migration
of a TLC spot relative to four calibrators on plate 1A
(see Detection reagents).

(d) This reagent, which detects barbiturates and several
neutral/weakly acidic drugs, is referenced on the data
cards but is not included in the computer program.

Table 2. TLC (a) visual detection limits
of several drug types determined with
SPOT ID reagents and techniques.

                                                         Detection
                                                          limit,
     Drug             Detection reagent, technique        ng/spot

Amphetamine       S1 366 nm, fluorescence                   10
Amitriptyline     S3 D1, visual observation                 100
Benzoylecgonine   S3 D1, visual observation                 50
Benzoylecgonine   S6 D2, visual observation                 25
Caffeine          S6 D2, visual observation                 200
Caffeine          S7 UV 254 nm, in viewing box              25
Carbamazepine     S8 [Cl.sub.2] vapor and 366 nm light       5
Desipramine       S2 FPN, lightly heat, blue color          20
Diphenhydramine   S4 Marquis, yellow color                  100
Lorazepam         S8 [Cl.sub.2] vapor and 366 nm light      20
Mesoridazine      S5 Mandelin, pink color                   20
Methampetmine     S1 366 nm, fluorescence quench            30
Morphine          S4 Marquis, purple color                  40
Nortriptyline     S4 Marquis, red-brown color               20
Phencyclidine     S3 D1, visual observation                 200
Phencyclidine     S6 D2, visual observation                 100

(a) Analtech no. 47521 scored silica-gel plates with
fluorescent indicator were developed to 10 cm with
the solvent system described in the text.

Table 3. TLC visual detection limits for several
drugs extracted from 0.5 mL of unbuffered serum.

                           Detection limit, mg/L

    Drug        UV 254 nm   [Cl.sub.2]   HCI    HgS[O.sub.4]

Acetaminophen     0.50         2.5        NR         NR
Caffeine          0.10          NR        NR         NR
Carbamazepine     0.20         0.02      0.04        NR
Carisoprodol       NR           NR        NR        1.0
Nordiazepam       0.10         0.05      0.1         NR
Theophylline      0.20          NR        NR         NR
Secobarbital       NR           NR        NR        0.2

NR, no obvious reaction with the reagent indicated.

Table 4. TLC visual detection limits for several basic and
neutral drugs recovered by liquid-liquid extraction from 5
mL of pH 9 buffered urine.

                           Detection limit, mg/L

Drug substance    UV 254 nm   Fluorescamine   FPN      D1

Amphetamine       Weak (a)         0.2        NR      Weak
Benzoylecgonine      0.2           NR         NR      0.15
Codeine              0.3           NR         NR      0.2
Desipramine          0.1           0.2        0.1     0.5
Diphenhydramine     Weak           NR         NR      0.5
Imipramine           0.1           NR         0.1     0.5
Mesoridazine         0.1           NR         0.1     0.4
Methamphetamine     Weak           0.1        NR      0.4
Morphine             0.3           NR          2      0.4
Nortriptyline        0.2           0.2        NR      0.4
Phencyclidine       Weak           NR         NR    0.15 (b)

NR, no obvious reaction with the reagent indicated.

(a) Weak reaction observed.

(b) Dragendorff (D1) oversprayed with iodinated Dragendorff (D2).

Table 5. Summary of SPOT CHEK computer program listings from
the input of SPOT ID reactions from seven typical patients.

                  Characteristic in program

Patient
  no.      Plates used   S1   S2   S3   S4      S5

   1           1B
               1B
             1B, 2A      -    +    +
           1B, 2A, 2B    -    +    +     -
   2           2A        -    -    +
             2A, 2B      -    -    +     9
           2A, 2B, 2C    -    -    +     9       9
   3           2A        -    -    +
             2A, 2B      -    -    -    11
   4           2A        -    +    +
   5           2A        Q    -    +
             2A, 2B      Q    -    +     5
   6           2A        Q    -    +
           2A, 2B, 2C    Q    -    +     -       4
   7           2A        -    -    +
             2A, 2B      -    -    +     6

                  Characteristic in program

                                              No. of
Patient                                       matches
  no.          S6        S7   S8   S9   S10     (a)

   1            +        +    +          3      44
                +        +    +    +     3      22
                +        +    +    +     3      11
                +        +    +    +     3     4 (b)
   2                     +               3      31
                +        +               3       2
                +        +               3     1 (c)
   3                     +               2      12
                +        +               2     2 (d)
   4                     -               3     1 (e)
   5                                     1       3
                +        +               1     1 (f)
   6                                     4       6
                +        -               4     1 (g)
   7                     +               5       6
                +        +               5     1 (h)

(a) Relative to the reactions entered. (-)
indicates a negative reaction and (+)
indicates a positive reaction; a blank means
the reaction was not entered; Q indicates
fluorescence quenching; under S4 and S5,
the color is given by the number (see code in text).

(b) Carbamazepine, lorazepam, nordiazepam, oxazepam.

(c) Dextromethorphan.

(d) Guaifenesin, methocarbamol.

(e) Erythromycin.

(f) Azacyclonol.

(g) Lidocaine metabolite (MEG-X).

(h) Amiodarone.
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Title Annotation:Drug Monitoring and Toxicology
Author:Siek, Theodore J.; Stradling, Corey W.; McCain, Marc W.; Mehary, Timothy C.
Publication:Clinical Chemistry
Date:Apr 1, 1997
Words:5804
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