High Performance Liquid Chromatography Method for the Determination of Dextropropoxyphene in Hair and Nail

 

Cijo John1*, Priyankar Ghosh2, K.M. Varshney2, S.K. Shukla1, S.Satyanarayana3

1Central Forensic Science Laboratory, Govt. of India, Chandigarh, India-160 036

2Central Forensic Science Laboratory, Govt. of India, Hyderabad, India- 500 013

3Department of Chemistry, Osmania University, Hyderabad, India- 500 007

*Corresponding Author E-mail: cijojohn01@gmail.com

 

ABSTRACT:

Dextropropoxyphene, a narcotic analgesic is one of the highly abused drugs in recent times.  An analytical methodology was developed for the detection and quantitation of dextropropoxyphene in hair and nail samples collected from drug addicts. The method involves the decontamination of hair and nail using 2ml each of 0.1% SDS (sodium dodecyl sulphate), water and methanol sequentially under ultrasonication of 3 minutes duration and followed by digestion with methanol. The digested samples were undergone Solid Phase Extraction (SPE) using normal silica stationary phase with acetonitrile as the eluent and the extracts were taken for HPLC analysis. The separation was achieved on a Phenomenex Gemini C18 HPLC reversed-phase column (150mmx4.6mmx5µ) with diode array detection with UV range 200- 370 nm and quantification carried out at 217nm. The calibration plot for the determination of dextropropoxyphene is based on linear regression analysis, y= 13.584x + 52.885 with a linear regression coefficient of r2= 0.9990. The limit of detection of dextropropoxyphene was estimated as 0.5µg/ml and the limit of quantification was estimated as 2µg/ml. The amount of dextropropoxyphene detected in the hair and nail was found to be in the range of 0.0433- 0.0383 µg/mg and 0.1828-0.0696 µg/mg respectively. The presence of dextropropoxyphene is confirmed by injection of the fraction collected from HPLC to a GC-MS system. The method is rapid and reliable for the qualitative and quantitative analysis of dextropropoxyphene in hairs and nails and it can be used by law enforcement laboratories for routine analysis.

 

KEYWORDS: Hair and nail analysis, Dextropropoxyphene, Drug abuse, HPLC, GC-MS.

 

 

INTRODUCTION:

Dextropropoxyphene {(α S)-α-[(1R)-2-(Dimethylamino)-1-methylethyl]-α-phenylbenzene-ethanol propanoate} which has been observed to be widely abused in India in recent times is a synthetic weak opioid and it is abused due to its effect on the central nervous system (structure given in Fig.1) By action it is an analgesic which is used to alleviate pain disorders. If it is mixed with alcohol, it can cause lethal conditions with the overdosage1.

 

Hair is being recognized as a third fundamental biological specimen for drug testing besides urine and blood. Due to sample stability at wide range of temperature for long period, easy sample collection and a wider time window of detection, hair as a sample has evoked keen interest among Forensic Toxicologists all over the world.

 

At present, hair analysis is used as a tool for detection of drug use in forensic science, traffic medicine, occupational medicine and clinical toxicology in a number of countries. The major practical advantage of hair testing compared to the urine or blood analysis is that it has a larger surveillance window [weeks to months, depending on the length of the hair shaft, against 2 to 4 days for most drugs in urine and blood]. Urine analysis and blood analysis provide short-term information of an individual’s drug use, whereas long-term history is accessible through hair analysis. While analysis of urine and blood specimens often cannot distinguish between chronic use and a single exposure, hair analysis can make the distinction. Hair may be used to back up disputed urine or oral fluid tests, particularly where an individual claims not to be a regular drug user and that, for example, his or her drink was spiked without their knowledge.2-5

 

The analysis of nails for detection of drugs has not received much popularity as hair analysis. Nails were used for the analysis of heavy metals like arsenic 6 and drugs like amphetamines7, sedatives and benzodiazepines 8 which came out with good results comparable with hair analysis. The studies demonstrated that drugs may appear in nail clippings within 1 month of ingestion which is similar to hair. Thus nails act as a useful specimen for confirming the results obtained through hair and increase the amount of sample available for the analysis.

 

Some workers reported on the analysis of dextropropoxyphene from urine and blood 9-11 and from hair10, 12 but no work have so far been reported on nail.

 

Fig. 1 Structure of dextropropoxyphene

 

The present work illustrates a method for the determination of dextropropoxyphene using High Performance Liquid Chromatography (HPLC) in hairs and nails .The method was developed and validated using pure standard reference material of the drug as per the International Conference on Harmonisation (ICH) guidelines13. The developed method was applied to actual samples of hair and nail collected from the reported drug addicts where dextropropoxyphene was successfully identified and quantified. In forensic context, the developed method will be highly significant as the studied drug is forensically important and encountered most often.

 

EXPERIMENTAL:

Chemicals and reagents

The standard reference material of dextropropoxyphene (purity > 99%) was provided by M/s Intas Pharmaceuticals Pvt.Ltd, Ahmedabad, India. The solvents: methanol, acetonitrile, water, hexane, 2- propanol (all HPLC grade) was procured from Qualigens fine Chemicals Pvt Ltd. India. Orthophosphoric acid, Sodium dodecyl sulphate and Potassum dihydrogen phospate was procured from Merck Specialities Pvt Ltd. India. The SPE cartridges (Agilent SAMPLIQ Silica with normal silica stationary phase) were provided by M/s Agilent technologies Pvt. Ltd.

 

Instrumentation

The HPLC analysis was carried out on a Shimadzu (Kyota, Japan) HPLC system consisting of an LC- 10 AT pump, an SPD-M10A PDA detector, an SIL- 10AD autosampler and a DGU-14A degasser  totally controlled by Class VP-10 software. The GC-MS analysis was performed on a Perkin Elmer Clarus 600S GC-MS system which is controlled by Turbomass 5.3.0 Software.

 

Sample collection

The hair samples were collected as per the guidelines given by the Society of Hair testing, France.14 Hair samples of two reported drug abusers  who were undergoing the de-addiction have been collected by cutting the samples from posterior vertex region of the head (where the hair growth is uniform) as close to the scalp as possible.  Nail samples of the same drug users have been collected by cutting the nails from both hands and legs.

 

Sample preparation

The collected hair and nail samples were weighed and the hair was taken about 200 mg each and nail was taken about 60 mg each. The samples were decontaminated using 2ml of 0.1%SDS (sodium dodecyl sulphate) solution followed by 2ml of water and 2 ml of methanol under ultrasonication for 3 minutes each. The samples were totally dried and the hair samples were cut into small segments of about 1mm each and the nail samples cut into small pieces as possible.

 

Extraction of the sample

The samples were taken in 2ml centrifuge tubes separately and 1 ml of methanol is added and kept for ultrasonication for a period of 4 hours. After that the solution is taken out and solid phase extraction is carried out using Agilent SAMPLIQ Silica cartridges with capacity 200mg/3ml. The conditioning was done by 2ml isopropanol followed by 2 ml Hexane. 200 µl of each of the samples were loaded and the washing was done with 1.5 ml mixture of 2% isopropanol in hexane (500 µl each three times). The elution was done with acetonitrile with a final volume of 1 ml.

 

HPLC analysis

A Phenomenex Gemini C18 HPLC column with dimensions (150mmx4.6mmx5µ) protected with a Phenomenex guard cartridge was used throughout the analysis. The mobile phase used was Acetonitrile: 50mM potassium dihydrogen phosphate (1:1 v/v), pH adjusted to 4.1 by addition of orthophosphoric acid with a constant flow rate of 1 ml/min. The chromatographic runtime was 7 minutes with a sample injection volume of 10 µl. The column oven temperature was maintained at 400C and the detector PDA range was 200- 370 nm and quantification was carried out at 217 nm.

 

Working standard reference solution

25 mg of standard reference material of dextropropoxyphene was accurately weighed and dissolved in the above said mobile phase solvent in a 25 ml standard flask which is used as the stock solution of concentration, 1mg/ml.

 

Method validation studies

Linearity and range

Different concentrations of the standard dextropropoxyphene ranging from 0.1ug/ml to 500 ug/ml were prepared and the chromatographic run with the above mentioned conditions for the same samples was carried out. 10 µl of each of the concentrations was injected to the HPLC system for the calibration studies.

 

 

The chromatogram was examined and the peak areas of the corresponding concentrations are noted at a wavelength of 217nm. A calibration curve of peak area against concentration is drawn to study the linearity and range of the developed method. The linear regression coefficient also is calculated from the calibration curve.

 

Precision

The precision of the method developed was examined by checking the repeatability of retention time (Rt value) and peak area of four different concentrations 300, 200,100, 50 µg/ml with two replicates of each of the concentrations. The intra and inter day precision was studied for the same concentrations and expressed in terms of percentage relative standard deviation.

 

Robustness

The robustness of the HPLC method was checked by making slight variations in pH of the mobile phase ( 4.05, 4.1, 4.15 with constant temperature at 400C and mobile phase ratio 1:1), composition of the mobile phase (1:1, 0.95:1.05 and 1.05:0.95 with constant pH at 4.1 and temperature at 400C)and temperature of the column oven (39, 40, 410C with constant pH at 4.1 and mobile phase ratio 1:1) and carrying out the analysis for a single concentration 300ug/ml. The Rt value and the peak area was noted for each of the analysis and the percentage relative standard deviation was calculated to study the robustness of the method.

 

Detection limits (LOD/LOQ)

The standard dextropropoxyphene was prepared in different lower concentrations coming down upto 0.1ug/ml and carried out the analysis to determine the limit of detection (LOD) and the limit of quantification (LOQ). Blank mobile phase was also injected and the signal to noise ratio was determined.

 

Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of standard with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected and quantified. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit whereas a typical signal-to-noise ratio of 10:1 is acceptable for quantification limit. [13]

 

Specificity

The specificity of the method was determined by comparing the analytical results given by the standard dextropropoxyphene and those by the hair and nail samples. The Rt values and the UV spectra of the standard and that of the samples are compared to obtain the specificity. The peak purity of the standard was assessed by comparing the spectra at three different levels, at peak start, at peak apex and at peak end of the corresponding chromatographic peak.

 

Quantification of dextropropoxyphene from hair and nail samples

The extract evaporated after SPE is reconstituted with 1 ml of mobile phase and taken for HPLC analysis. 10 ul each of the samples were injected during the chromatographic run. The chromatograms obtained for each of the sample were integrated at a wavelength of 217 nm for quantification. The calibration curve drawn with the external standard was used for estimating the concentration of dextropropoxyphene from the corresponding hair and nail samples.

 

Confirmation by GC-MS analysis

The fraction of the mobile phase corresponding to the peak is collected, evaporated to dryness, re dissolved  in methanol and injected to the GC-MS for further confirmation. The GC column used for the analysis was PE Elite- 5MS column with dimensions 15m x 0.25 mm x 0.1 µm. The detection carried out by mass spectrometer with EI (Electron Impact) ionisation and   was monitored in full scan mode with a mass range of 40 to 600 amu. A solvent delay of 1.5 minutes was given during the run to avoid the solvent.

 

RESULTS AND DISCUSSION

HPLC analysis

The HPLC analysis was carried out as discussed above and the chromatogram obtained for standard dextropropoxyphene is given in Fig.2. The retention time was found to be 2.496 minutes. The UV spectra recorded at the retention time shows that the lambda maximum is 256nm for the standard. The purity of the chromatographic peak is determined by the taking the spectra at different points of the peak (from peak start to peak end) and overlaying the different spectra obtained. The overlaid spectrum shows the purity of the standard is acceptable.

 

Fig. 2 HPLC Chromatogram of Standard dextropropoxyphene  

 

Method validation studies

Linearity and range

The calibration curve (plot of peak area versus concentration) for dextropropoxyphene was drawn (Fig.3) as per the data obtained for various concentrations ranging from 0.5ug/ml to 400 ug/ml, analysed using the HPLC method.

 

 Fig. 3 Calibration curve of standard dextropropoxyphene.

 

The linear regression equation obtained from the calibration curve is given by,

y = 13.584x + 52.885 with a linear regression coefficient, R2= 0.9990 which clearly shows the excellent linearity of the developed method. The range of linearity of dextropropoxyphene is found to start from 0.5ug/ml onwards below which it is not detectable and up to 400ug/ml above which the linearity decreases. In other words dextropropoxyphene is found to be linear in the concentration range of 0.5 to 400ug/ml.

 

Precision

The precision of the developed method was examined by carrying out the repeatability of the method with different concentrations both intraday and inter day. The mean peak area as well as retention times recorded each time was taken and the standard deviation and the % RSD calculated which is given in the Table 1 and Table 2 respectively. The lower values of %RSD (0 to 4%) shows the good precision of the method.

 

Robustness

The robustness of the method was checked by making slight variations in conditions like mobile phase pH, composition and temperature of the column oven. The method was found to be robust as the slight variation in different parameters does not make significant deviation in the values of peak area and retention time for the particular concentration, 300 µg/ml (Table 3). The lower values for %R.S.D makes it clear that the method is robust enough to carry out the analysis of dextropropoxyphene.

 

Detection limits (LOD/LOQ)

The Limit of detection and Limit of quantification for dextropropoxyphene using the developed method was determined using the signal to noise ratio method. A concentration of 0.5ug/ml was found to be the detection limit and the quantification limit was found to be 2 ug/ml. The lower values of LOD and LOQ suggest the method is sensitive enough to detect and quantify the analyte in trace amounts.

 

 

Table1: Data for precision studies with respect to peak area.

Concentra-tion ug/ml

Mean area (AU)

Standard deviation

% R.S.D

Inter day

Intra day

Inter day

Intra day

Inter day

Intra day

50

729446.5

727634

2257.79

305.47

0.3095

0.0419

100

1335749

1335980

697.20

371.23

0.0521

0.0277

200

2590299

2584806

6616.39

1151.87

0.2554

0.0445

300

3665701

3664561

3161.47

1549.27

0.0862

0.0422

 

Table 2: Data for precision studies with respect to retention time.

Concentration ug/ml

Mean retention time

Standard deviation

% R.S.D

Inter day

Intra day

Inter day

Intra day

Inter day

Intra day

50

2.4265

2.496

0.0983

0

4.05

0

100

2.4265

2.496

0.0983

0

4.05

0

200

2.432

2.5015

0.0905

0.0077

3.72

0.3109

300

2.432

2.5015

0.0905

0.0077

3.72

0.3109

 

Table 3: Data for robustness with respect to peak area and retention time.

Parameter

Mean peak area

Standard Deviation

% R.S.D

Mean retention time

Standard deviation

% R.S.D

Temperature  (39, 40,41)0C

3647613

56353.31

1.50

2.513

0.0162

0.6468

Mobile phase pH (4.05, 4.1, 4.15)

3663806

1880.19

0.0513

2.485

0.1123

4.5217

Mobile phase ratio(1:1,0.95:1.05 and 1.05:0.95)

4097078

57096.53

1.39

2.531

0.1186

4.6867

 

Quantification of dextropropoxyphene from samples and Specificity

The chromatograms obtained during the HPLC run for one of the samples is given in Fig.4 and Fig.5

 

Fig.4 HPLC Chromatogram for sample 1, Hair

 

Fig.5 HPLC Chromatogram for sample 1, Nail

 

The amount of dextropropoxyphene present in the samples was calculated from the calibration curve of standard dextropropoxyphene. The corresponding amount is given in Table 4 for each of the samples analysed.

 

Table 4: Data regarding the quantification of dextropropoxyphene from samples.

Samples

Amount in µ g/ml

Amount in µ g/mg of (hair/nail)

Sample 1, hair

10.828

0.0433

Sample 1, nail

9.140

0.1828

Sample 2, hair

7.652

0.0383

Sample 2, nail

5.573

0.0696

 

The method was found to be highly specific for dextropropoxyphene as the chromatographic peak obtained for the samples have identical retention time with the standard and have good resolution clearly separating other components present. The UV spectra obtained with the samples was perfectly matching with that of the standard with the lambda max 256 nm. The purity of the peak also was checked and found to be excellent. All the results suggest the developed method is highly specific.

 

The amount of dextropropoxyphene found in hair and nail of both the abusers was found to be in trace quantities, in nanogram level. In both the cases the amount of the analyte found in nail was larger than that in hair which is a matter to be explored further. The drug incorporation mechanism and rate depends upon various factors like the chemical property of the compound, metabolic activity, amount and frequency of drug intake etc. which needs further study.

 

Confirmation using GC-MS

The GC-MS chromatograms of the standard and sample fractions collected during HPLC run is given in Fig: 6 which clearly indicate the presence of dextropropoxyphene in both the sample and standard at retention time 4.51 minutes.

 

Sample

Fig 6: GC-MS chromatograms of standard and sample.

 

 

The results obtained clearly suggest the method is highly sensitive enough to detect the presence of dextropropoxyphene from the biological matrices, hair and nail. Also the digestion and extraction procedure adopted was good enough to bring out the incorporated analyte from the complex protein matrix of hair and nail.

 

The drug abuse history of the subjects undergone study were more than five years and the prevalence of the original drug in both hair and nail confirms the abuse by the individuals. As dextropropoxyphene is a scheduled drug under Narcotic Drugs and Psychotropic Substances Act, the analytical methodology developed will help the forensic toxicologists as well as the various law enforcing agencies to a great extend in supporting the criminal justice system.

 

The results establish that drugs are incorporated into growing nails at levels comparable to those in hair. Analysis of nail clippings can therefore be used either alone or preferably in complementing hair analysis to establish ingestion of drugs.

 

CONCLUSIONS:

The analytical method reported in this paper for the estimation of dextropropoxyphene from hair and nail using HPLC is found to be very sensitive, robust and a precise one which can be routinely used in Forensic Laboratories encountering cases related to the same analyte.  The solid phase extraction method adopted was found to be good enough to give acceptable results. Hair and nail which are unusual biological specimens for analysis is proved to be very powerful tools to identify the analyte when conventional specimens like blood and urine fails or it can be used as a confirmatory test for the presence of the analyte.

 

ACKNOWLEDGEMENT:

The authors are highly thankful to the Chief Forensic Scientist, Directorate of Forensic Science Services, New Delhi for providing the fellowship to carry out the research work. Also the authors are thankful to Shri. A.K. Ganjoo, Director, CFSL Hyderabad for providing the facilities to carry out the research work.

 

REFERENCES:

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Received on 05.06.2012        Modified on 02.07.2012

Accepted on 18.07.2012        © AJRC All right reserved

Asian J. Research Chem. 5(8): August, 2012; Page 966-971