Detection of Alkaloid Abuse Drugs in Liquid Samples by Clinical Laboratory Methods

Aim: Fifty fluids sample of abusers were examined for the presence of alkaloid abuse drugs in body liquids. Abuse drugs of liquid samples directly confirmed with rapid strip tests and their extracts were detected by quality and quantity TLC- UV methods.All of the study population were male and their age range were (Mean ±SD) = (41±22). 

Methods: Samples were daily collected from abusers of Tabriz/Iran. Urine and Saliva extracts were prepared by using LPE method. The LPE were made and shacked by 9% saline solution and organic solvents of methanol and pure ethanol and cholorophorm and ammoniac with base productive of Merck company for every 10ml of liquid samples. Then after centrifugation under concentrated section of tubes were extracted. After separation to dryness with heater, the residue was dissolved in organic solvent of methanol to reach 20 to 50-fold concentration. For TLC-UV process, the extraction residues were provided to spotting on TLC plates for doing of thin layer chromatography. So, the detection limit of different abuse drugs standards was between 300-750 ng/ml. 

Results: The quantity amount data analyzed by SPSS and IMAGEJ software’s. The result of this study has shown that above 94 percent of liquid samples from abusers in welfare organization of Tabriz /Iran were contained alkaloid abuse drugs and above 25 percent of these samples have shown drug interaction. Algorithm of constant Retention Factor parameter (RF) were comparisoned to all standards and abuse drugs. 

Conclusion: This study describes importance of liquid phase extraction (LPE) and drug testing such as TLC analysis in comparison of other rapid tests.

Nowadays abuse drugs are globally problems that make one of the most side effects to public health. So, the selection of achievement methods to therapeutic drug monitoring (TDM) are requirement. The recovery related policy and guidelines issues not enough about the nature and distribution of all abuse drugs in the world. The perfected data to prevention and treatment of abuse disease are necessary, depend on different regional and globalization. The geographic local of some countries makes easy transit of opium compounds to the other west countries. Possibility of clinical positions to diagnostic and treatment of addiction are different in Middle east countries such as Iran. So, this study routinely refers to abuse drugs diagnosis in fluid samples such as urine and saliva. All abuse drugs and their metabolites are detectable in liquid samples. Then we have done rapid tests and confirmed of them by TLC method. Moreover, some of abuse drugs might be distinguished in these studies such Diacetyl morphine, Ethyl morphine, Hydromorphoine, Apomorphine, Morphine, Cannabis, Methadone, Tramadol, Buprexin, Amphetamine, Methamphe-tamine, Methylene Dioxy methamphetamine, Cocaine, Phencyclidine, Benzodiazepine, Three cyclic antidepressant, Phenobarbital and etc.., through this method.  So, this epidemiological study supported by the clinical laboratory of welfare organization and Hygiene center. The quantities data related on detection not enough about the nature of all kinds abuse drugs in the world. These methods are extremely helpful in quick diagnosis of abuse compounds. So, several studies including screening and confirmation of abuse drugs by thin layer chromatography have done by TLC-UV system in forensic laboratories. So, this study exactly shows quality and quantity importance of thin layer chromatography for detection of drugs and abuse drugs by application of ImageJ software. With this background this study carried out TDM (Therapeutic Drug Monitoring) with TLC-UV system at toxicology laboratories of welfare organization and hygiene center in Tabriz/Iran [1-7].

Samples providing

2ml of every sample reached to 5ml with saline solution and one droplet of ammoniac to providing liquid phase extraction process (LPE). First some of samples were directly examined for presence or absence of some drugs and abuse compounds such morphine, amphetamine, methamphetamine by rapid strip tests. Each addicted subject was asked to give a fresh sample of his saliva and urine to the laboratory before recovery. All study population filled satisfy form to participating in study. Besides providing samples, agreed to complete a questionnaire focusing on their conditions and abuser history and life style conditions characteristics. All of participants were male mean aged were 41±22 years. All of them were smokers too. The study was submitted and approved by the local ethical committee. All gave their informed consent prior to them in the study. All liquid samples were daily collected from abusers and stored in refrigerator, then pH of samples changed to alkaline condition with adding one ammoniac droplet. The pH regulated with pH paper for each sample. Extraction tubes were provided with 1 ml pure 96% Merck company ethanol and 2ml of cholorophorm and 2ml methanol and 5ml of samples.

Rapid Tests

First, all liquid samples were examined with rapid strip tests then all urine and saliva extracts were examined with TLC method. However, the results of saliva and urine strip tests were positive before recovery.

LPE and TLC-UV process

All samples were daily collected from abusers of Tabriz/Iran. Urine and Saliva extracts were prepared by using LPE method. The LPE were made and shacked by 9% saline solution and organic solvents of methanol and pure ethanol and cholorophorm and ammoniac with base production of Merck company for 5ml of liquid samples. Then after centrifugation under concentrated section of tubes were extracted. After separation to dryness with heater, the residue was dissolved in organic solvent of methanol to reach 20 to 50-fold concentration. For TLC-UV process, the extraction residues were provided to spotting on TLC plates for doing of thin layer chromatography. So the detection limit of different abuse drugs standards was between 300-750 ng/ml. After the TLC process for confirming of spots tests for the all alkaloids were comparisoned with standards spots and were visualized by UV cabinet. [3-18]. In first step of this study all samples with adding one droplet ammoniac were directly done with rapid tests then confirmed with TLC- UV methods. After doing of TLC, quantity of abuse drugs by area under the curve (AUC) and IMAGEJ software were determined. 

AUC graphs and data of this study were analyzed by IMAGEJ software. According to results of this study Table1: Shows number of drugs and abuse drugs consumption lists between the study group before recovery.

Table2: Shows some of morphine TLC tests and standard quantities in before and after recovery. Table3: Shows some of amethamphetamine TLC test and standard quantities in before and recovery. Table4: Shows some of methamphetamine TLC test and standard quantities in before and recovery. Table5: Shows some of TLC test results that have shown drug interaction after recovery. Figure1 and Figure2 and Figure3 show AUC quantity of amphetamine and methamphetamine and morphine standards. Also, Figure4 shows AUC quantity of morphine TLC test in one patient. Image1.shows TLC plate with content negative results of amphetamine and methamphetamine in one recovered patient. Also, this plate shows that the RFof amphetamine(left) is more than the RFof methamphetamine(right)standard spots. Image2. shows TLC plate with content positive results of morphine(left) and morphine and codeine standard (right) in one unrecovered patient. Also, this plate shows that the RFof codeine (up right) is more than the RFof morphine (down right) standard spots. The RF (Retention Factor) is constant parameter for every drug in thin layer chromatography tests. The RF value indicates the relative distance one substance travels compared to the solvent. Hereby a ratio calculated in TLC representing the distance travelled by a component divided by the total distance travelled by the solvent. All calculation data was performed using SPSS and IMAGEJ softwares. The data were analyzed by using of variance (ANOVA ONE WAY) by post hoc comparison between groups were examined with student Newman kelus test and t-test. Differences in which p<0.05 were considered statistically significant. The results of this study show that above 94 percent of liquid samples from opioids abusers in Tabriz /Iran were contained alkaloid substances. however, drug interaction was happened in above 25 percent of these samples. [1-20].

Figure 1: AUC of amphetamine standard dose

Figure 2: AUC of methamphetamine standard dose

Figure 3: AUC of morphine standard dose

Figure 4: AUC one of patient morphine TLC test

Image 1: shows TLC plate with content negative results of amphetamine(left) and methamphetamine(right) in one recovered patient.

Image 2: shows TLC plate with content positive results of morphine(left) and morphine and codeine standard (right) in one unrecovered patient.

Table 1: Shows number of drugs and abuse drugs Consumption lists between the study group before recovery.

N=50, BNZ: Benzodiazepines, TCA: Three cyclic antidepressants, BAR: Barbiturates, TRM: Tramadol, PCP: Phencyclidine, THC: Cannabis, COC: Cocaine. Urine samples, Saliva fluid samples. 

Table 2: Some of Morphine TLC tests and standard quantities in before and recovery period.

N=25, TLC test results Morphine and standard quantities (STD conc) in men abusers, Urine extract samples. Weak positive (75ng/ml<30 p=0.01,><05. Urine samples

Table 3: Some of A methamphetamine TLC test and standard quantities in before and recovery period.

N=25, TLC test results Amethamphetamine and standard quantities (STD conc in men abusers, Urine extract samples. Weak positive (50ng/ml<30 p=0.005,><05. Urine samples

Table 4: Some of Methamphetamine TLC test and standard quantities in before and recovery period.

N=25, TLC test results methamphetamine and standard quantities (STD conc) in men abusers, Urine extract samples. Weak positive (30ng/ml<30 p=0.004,><05. Urine samples

Table 5: Some of TLC test results have shown drug interaction after recovery process of some cases.

N=50, Drug interactions (Drug dose) have after recovery through TLC (Thin layer chromatography) method. Ages mean>20, N=15, BNZ: Benzodiazepines, TCA: Three cyclic antidepressants, BAR: Barbiturates, TRM: Tramadol.PCP: Phencyclidine-THC: Cannabis-COC: Cocaine. Urine and Saliva extract samples. Tests comparison with their standards.