Automated Solid Phase Extraction Method for Testing Forensic and Toxicology Samples

Due to their biological complexity, toxicology and forensic samples are not suitable for direct analysis using ultra performance liquid chromatography-mass spectrometry (UPLC®-MS). In order to obtain accurate analytical results, an effective sample preparation and extraction technique is required.

It would be ideal if the sample preparation technique is selective, fast, accurate, safe, and cost-effective. However, the usual manual methods are labor-intensive and prone to human error, reducing turnaround times, lowering productivity, and increasing costs. Quality compliance (ISO 17025) becomes an increasingly difficult task due to lack of data traceability.

This article explains a study that was conducted to develop an automated process for handling forensic samples, extracting 23 opioids, cocaine, and their metabolites from human urine, and meeting the following criteria:

  • to reduce hands-on time by over 50%
  • to minimize sample cross-contamination
  • to integrate with Laboratory Information Management Systems (LIMS)
  • to monitor all sample transfer and extraction steps

For this purpose, researchers employed a Tecan Freedom EVO® positive pressure solid phase extraction (SPE) workstation for complete automation of sample extraction, right from urine tubes through to vials ready for UPLC-MS analysis. A UPLC-MS system was then used to analyze the extracted samples.

The workstation is capable of automating liquid transfer steps, such as addition of internal standards, handling urine, quality controls and blanks, and even sample hydrolysis (if necessary) and SPE steps.

The technique was validated for selectivity, precision, accuracy, recovery, and matrix effects, employing authentic samples as well as those from proficiency testing schemes containing heroin, cocaine, methadone, and buprenorphine.

Material and methods

Instrumentation

A Freedom EVO positive pressure SPE workstation was employed to carry out sample preparation. Figure 1 shows the specific components used to configure the workstation, using a Freedom EVO 100 platform with the additional components given below:

  • an eight-channel liquid handling (LiHa) arm configured with four disposable tips and four fixed needles
  • an SPE manifold for holding 32 SPE cartridges (3 ml, 60 mg)
  • a pick-and-place arm (PnP) for automated tube handling
  • carriers for urine samples, SPE reagents, internal standards (IS)
  • a tube holder (Sommer Automatic)
  • a 1D/2D barcode scanner

Figure 1. Photo of the Tecan Freedom EVO positive pressure SPE workstation. The image shows the configurable parts of the worktable used by researchers for this study.

Materials

In this study, individually certified reference standard solutions were procured from LGC standards (Molsheim, France). Methanol (UPLC-MS grade), water (HPLC grade), and 0.1% formic acid were bought from Biosolve (Valskenswaard, NL).

VWR (Leuven, Belgium) provided β-glucuronidase from Helix pomatia. Strata-XTM cation exchange cartridges were obtained from Phenomenex (Utrecht, NL) and Oasis® MCX cartridges (3 cc, 60 mg) from Waters (Zellik, Beligum).

Workflow

The workflow given below was implemented on the Freedom EVO® cartridge SPE workstation.

Since 6-monoacetylmorphine (6-MAM) may be degraded by β-glucuronidase, the hydrolysis step was performed only in specific cases. The steps which are in blue color were performed on the Freedom EVO.

The incubation was done offline as it is a long, overnight step. Buffer 1 in the workflow is 675 μl of sodium acetate buffer 0.1 M (pH 4) or 675 μl of water (for hydrolyzed samples). Buffer 2 is 1 ml ammonium formate buffer 5 mM (0.05% formic acid). The solvent compositions used for the various SPE steps are:

  • Conditioning – 1 ml methanol followed by 1 ml sodium acetate buffer
  • Washing – 0.8 ml 0.1 N HCl followed by 0.8 ml methanol
  • Eluting – 1.5 ml dichloromethane: 2-propanol:ammonia (80:18:2, v/v/v)

Figure 2. Workflow

The air push volume in between successive SPE steps was carefully optimized, so that the columns are dried after every step. This is important to prevent phase immiscibility that can lead to inefficient extraction, and prevent column overload with solvents.

Chromatographic conditions

Table 1. Chromatographic conditions used for the study

Analytical column

Acquity UPLC BEH phenyl (2.1 x 100 mm, 1.7 μm) (Waters).

Temperature

50 °C

Flow rate

0.35 μl/minutes

Solvents

Solvent A – 0.1% formic acid in water

Solvent B – 0.1% formic acid in methanol

Gradient

0-1.5 minutes – 5% solvent B

1.5-4 minutes – 25% Solvent B (linear increase)

4-7.5 minutes – 50% solvent B (linear increase)

7.5-9 minutes – 95% solvent B

Total run time

9 minutes

 

Mass spectrometry conditions

Table 2. MS conditions used for the study

Instrument

Quattro Premier (Waters)

Ionization

ESI+

Capillary voltage

1 kV

Source temperature

120 °C

Desolvation gas flow

900 l/h

Desolvation temperature

350 °C

Collision gas pressure

0.35 Pa

 

Multiple reaction monitoring (MRM) transitions

Table 3. MRM transitions and conditions for the 23 compounds and their ISs.

Compound

Precursor ion (m/z)

Product ion (m/z)

Morphine

286.0

164.9

200.9

Hydromorphone

286.0

157.0

185.0

Oxymorphone

302.0

227.0

284.0

Norcodeine

286.1

215.2

225.2

Codeine

300.1

183.4

215.2

Dihydrocodeine

302.1

171.2

199.1

Oxycodone

316.1

241.2

298.0

6-MAM

328.1

165.1

211.2

Hydrocodone

300.1

171.1

199.1

Ethylmorphine

314.1

157.2

165.1

Norfentanyl

233.1

84.0

150.0

Benzoylecgonine

290.1

104.9

168.0

Tramadol

264.1

58.2

246.3

Meperidine

248.1

174.0

220.1

Cocaine

304.0

82.0

181.9

Pentazocine

286.1

69.2

218.2

Cocaethylene

318.1

82.1

196.1

Fentanyl

337.1

105.0

188.3

Norbuprenorphine

414.1

82.9

100.9

EDDP*

278.1

186.1

234.1

Buprenorphine

468.1

54.9

84.0

Propoxyphene

341.0

58.4

266.3

Methadone

310.1

105.0

265.2

Morphine-d3

289.0

201.0

Codeine-d6

306.1

165.0

Dihydrocodeine-d6

308.1

202.1

6-MAM-d6

334.1

165.0

Benzoylecgonine-d8

298.1

171.0

Tramadol-d3

268.0

58.2

Meperidine-d4

252.1

178.2

Cocaine-d3

307.1

185.0

Fentanyl-d5

342.1

105.0

EDDP-d3

281.1

234.2

Buprenorphine-d4

472.1

59.3

Methadone-d9

319.1

105.0

 

*2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine

Method validation

Selectivity and specificity

In order to evaluate the method selectivity and specificity, chromatograms of healthy patient urine samples, both spiked and non-spiked with the IS, were compared. Also evaluated were authentic urine samples from drug users (amphetamines, cannabis, and benzodiazepines).

Linearity, limit of quantitation, precision and bias

Two sets of urine calibrators were arranged in 0.1 M sodium acetate buffer, with the Group 1 having a concentration range of 750, 500, 250, 125, 75, 50, 25, and 15 ng/ml, and Group 2 having a concentration range of 150, 100, 50, 25, 15, 10, 5, and 3 ng/ml.

Then, standard curves were prepared with every batch of QC and authentic samples, by employing least squares quadratic or linear regression, with a weighting factor of 1/x for all the compounds.

To determine limits of quantitation (LOQ), replicate analysis (n = 2) over a period of eight days was evaluated. The LOQ was accepted if the calculated concentration was within 20% of the real value and had an RSD of <20%.

Intra- and inter-day imprecision was evaluated by analyzing QC samples in duplicate for eight days. The mean of the measured concentrations for the QC samples was compared with the nominal values to determine the bias of the method.

Matrix effects

Standard compounds were added to the blank urine samples to bring them to different concentrations, and the samples were subjected to automated SPE.

A comparison was made between the resulting peak areas/response and those obtained after spiking the mobile phase manually at the same concentrations. This was done in order to assess the matrix effects on ionization. For each concentration (n = 8), all analyses were performed in replicate.

SPE recovery

Non-deuterated compounds and ISs (deuterated compounds) are added to blank urine samples to bring them to different concentrations. Tests were performed twice, once by adding the non-deuterated compounds before automated SPE extraction, and then by adding them after extraction.

Finally, the deuterated compounds/IS peak area ratios were examined for the two situations to investigate the SPE recovery. All tests were performed in replicate (n = 8).

Cross-contamination

High concentrations of standards were used to spike blank urine samples, which were extracted followed by samples with IS only, so as to check for carryover. Authentic samples were used to intersperse blank samples, which were again extracted and analyzed to check for cross-contamination effects.

Carryover in the LC-MS system

After being spiked with 10x higher concentration than that of the LOQ of the standards, blank urine samples were injected in triplicate and then by a sample spiked with IS only, to evaluate carryover in the LC-MS system.

Results

The extraction and analysis method described above was confirmed for linearity, selectivity, imprecision, LOQ, bias, recovery, matrix effects, and carryover by employing international guidelines. Once validation was done, the method was used on authentic samples.

Analysis of blank urine samples followed by spiking of the urine samples with IS did not show any interference.

For Group 1 compounds, the standard calibration curves ranged from 15 to 750 ng/ml, and for Group 2 they ranged from 3 to 150 ng/ml. An RSD <20% was noted for all the compounds at the LOQ after automated sample preparation. The inter- and intra-assay precision were found to be satisfactory, with RSD <15% for all the compounds. According to the results, the bias of the assay was <5%.

Table 4. Extraction efficiency and RSD (%) at two concentration levels (n = 8 at each conc.). QC low and QC high are 30 and 600 ng/ml respectively, for all the compounds except fentanyl, hydromorphone, oxycodone, hydrocodone, norfentanyl, 6-MAM, buprenorphine, and norbuprenorphine (concentrations at 6 and 125 ng/ml).

Extraction efficiency

 

QC low

RSD (%)

QC high

RSD (%)

Morphine

89.6

3.5

82.7

1.8

Cocaine

89

3.9

91.6

1.9

Benzoylecgonine

83.3

5.1

87.2

3.7

Buprenorphine

100.3

7.2

79.5

2.6

Norbuprenorphine

91.9

10.1

69

10

6-MAM

72.3

2.7

77.3

4.9

Codeine

88.7

3.3

87.6

3.7

Hydromorphone

83.3

4.7

84.6

3.6

Hydrocodone

83.9

4.7

84.4

4.1

Norcodeine

82.5

4.9

81.7

1.9

Oxycodone

86.6

6.7

89

4.4

Oxymorphone

86.1

3.8

91.4

3.6

Dihydrocodeine

89.3

3.7

89.8

2.5

Ethylmorphine

90.2

3.3

86.1

3.1

Cocaethylene

91.4

4.2

89

3.7

Methadone

83.3

3.5

82.6

1.6

EDDP

75.4

4.6

71.2

4.2

Fentanyl

87.1

4.1

84.8

1.7

Norfentanyl

84.4

3.4

87.7

2.4

Tramadol

87.8

3.8

89.2

2.9

Pentazocine

84.2

7.1

75.3

8

Propoxyphene

88.4

10.3

70.8

10

 

Sample cross-contamination was not observed during automated processing of samples on the Freedom EVO®. This was also the outcome of using only fixed tips for applying positive pressure to the SPE cartridges and using disposable tips for sample pipetting.

The same automated SPE method was used to analyze the samples obtained from proficiency testing schemes (GTFCH and LGC) which resulted in z scores of less than 0.71. This article does not show detailed data from the proficiency testing schemes; for details, please refer to the article by Ramirez Fernandez et al. (1)

Conclusion

This investigation presents the effective development and validation of an automated hydrolysis (if necessary) and SPE extraction method on a Freedom EVO cartridge SPE workstation for the LCMS analysis of 23 opioids, cocaine, and other metabolites.

After its successful application to proficiency testing schemes, the method is now routinely employed for the quantitative determination of cocaine, opioids, and metabolites in urine in forensic toxicology cases. The investigation outlines the benefits of automation for toxicology/forensic analysis.

Pipetting accuracy

Pipetting errors are highlighted with the use of pressure monitored pipetting (PMP) on the liquid handling channels, thus increasing pipetting accuracy, as observed in the results (RSD for 10 μl <2.5%). There is no need to make any concentration correction.

SPE efficiency

Positive pressure is separately applied through individual liquid handling channels to each sample, so as to allow much greater control (flow rate) than a vacuum station. This yields reproducible SPE extraction with negligible dead volumes.

Time savings

The technique offers an end-to-end process automation solution, right from urine tubes through to LC-MS-ready samples in vials. On the whole, the time needed for sample preparation was reduced by more than 80% as compared to the manual process (e.g. other manual tasks can be done in parallel), removing the time constraints related to extraction.

Data traceability

Barcode usage enables the instrument control software (Freedom EVOware) to employ LIMS- generated worklists to direct particular SPE extraction sequences.

Error reduction

Automatic sequence generation based on pipetting accuracy, LIMS worklists, and an efficient SPE process substantially reducing repeat tests, human error, and eventually laboratory costs. Limiting direct contact of the technicians with chemicals and specimens also improves their safety, restricting their exposure to potentially risky materials.

ISO 17025 compliance

Data traceability features, together with the ability to monitor all sample movements and record the data, is of special interest to labs working in an ISO 17025-compliant set-up.

Costs

On the whole, reduction in solvent consumption, the substantial reduction in errors, and improved productivity together has considerable cost implications for toxicology laboratories.

Acknowledgement

The data for this study was generated and kindly provided by Dr Maria del Mar Ramirez Fernandez from the Federal Public Service Justice, National Institute of Criminalistics and Criminology, Belgium.

Reference

  1. Validation of an Automated Solid-Phase Extraction Method for the Analysis of 23 Opioids, Cocaine, and Metabolites in Urine with Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry María del Mar Ramírez Fernández, Filip Van Durme, Sarah M.R. Wille, Vincent di Fazio, Natalie Kummer and Nele Samyn Federal Public Service Justice, National Institute of Criminalistics and Criminology, Chaussée de Vilvorde 100, 1120 Brussels, Belgium J Anal Toxicol (2014) 38 (5): 280-288.

About Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Jul 14, 2018 at 7:11 PM

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