This Application Note was written by Dr. Bryn Flinders of the Dutch Screening Group, Gaetano Martinolaan 85, 6229 GD, Maastricht, The Netherlands, in collaboration with HTX Imaging.

Application & Background

Hair testing is a powerful tool routinely used for the detection of drugs of abuse in toxicology and forensic applications (1). The analysis of hair is highly advantageous as it can provide prolonged detection and chronological information about drug intake or chemical exposure in contrast to the analysis of biological fluids (2). However, current methodology involves complex and time-consuming sample preparation followed by liquid chromatography coupled with mass spectrometry (LC-MS). These techniques also require large amounts of hair that is lost during sample preparation. Techniques such as matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) have been extensively used for the detection of drugs and metabolites in tissue sections and are increasingly being explored for monitoring the distribution of drugs and metabolites in single hair samples (3,4). The aim of the work reported is to use the HTX TM-Sprayer to homogenously apply MALDI matrix onto longitudinally sectioned hair samples, followed by MALDI-FTICR-MS imaging to monitor the distribution of cocaine and its metabolites, as well as the anti-seizure medication carbamazepine and its metabolites, in single hair samples.

Experimental

Sample Preparation

Hair samples were first washed twice with dichloromethane (10 mL) to remove hair products, sweat and trace amounts of drug material. Longitudinal sections of hair samples were prepared using the previously reported method (5). The hair samples were inspected via microscopy to determine if the hair sample had been successfully cut. The sectioned hair samples were then transferred and mounted onto ITO coated glass slides with double-sided copper tape.

Matrix Deposition

The hair samples coated with 15 mg/mL DHB in 50:50 methanol:water with 0.2% TFA using the HTX TM-Sprayer (Figure 1B). It was found that multiple passes with a less concentrated matrix was necessary to extract the compounds from the hair. The samples were coated using the following parameters:

 
Table 1.  Spray parameters for hair samples.

Table 1. Spray parameters for hair samples.

 

MALDI Mass Spectrometry Imaging

Images were acquired using a Bruker SolariX (Bruker Daltonics) equipped with a 9.4T superconducting magnet. The instrument was operated in positive ion mode and continuous accumulation of selected ions (CASI) mode. The instrument was calibrated with red phosphorus prior to analysis. The mass range was m/z 100-500 with a CASI window focused around m/z 304 (cocaine) and m/z 237 (carbamazepine) with an 80 Da isolation window. The spatial resolution was 100 × 20 μm (cocaine) and 200 × 20 μm (carbamazepine), due to the lengths of the hair samples.

Results

Inspection of Hair Sections and Matrix Coverage

Following preparation the hair samples were inspected via microscopy, to determine if the hair sample had been successfully cut and transferred onto the glass slide. The optical image of the hair following sectioning (Figure 1A) shows the external cuticle, the fibrous cortex where the drug is bound and the medulla core. The image of the hair sample following matrix application (Figure 1B) shows an homogenous crystal coverage over the surface of the hair sample.

Figure 1.  Optical images of hair samples before and after matrix deposition. (A) Longitudinal sectioned hair sample prior to matrix application showing the center of the hair sample. (B) Hair sample following matrix application showing the homogenous crystal coverage.

Figure 1. Optical images of hair samples before and after matrix deposition. (A) Longitudinal sectioned hair sample prior to matrix application showing the center of the hair sample. (B) Hair sample following matrix application showing the homogenous crystal coverage.

MALDI-FTICR-MS Imaging of Cocaine and Metabolites in User Hair Samples

Once the optimal matrix solution and spraying parameters were found, hair samples from a cocaine user were analyzed. The MALDI-FTICR-MS images showed the distribution of benzoylecognine at m/z 290.1369 (Figure 2B), cocaine at m/z 304.1536 (Figure 2C) and cocaethylene at m/z 318.1627 (Figure 2D). By monitoring the presence of unique metabolites such as cocaethylene, a metabolite formed in the liver during the simultaneous consumption of cocaine and alcohol, confirms the detected drug occurs from actual ingestion. The results also correlated well with previous results obtained from the same samples.6 The images also demonstrate consistent and heavy abuse of cocaine. The length of the hairs is approximately 1.5 cm, which based on the average growth of hair (1 cm/month), corresponds to 1.5 months of growth. The spatial resolution across the length of the hair is 100 μm, which means each pixel corresponds to approximately 7 hours of growth.

Figure 2.  MALDI-FTICR-MS imaging of cocaine user hair samples. A) Optical image of longitudinally sectioned hair samples following matrix application. B) MALDI-FTICRMS images showing the distribution of B) benzoylecognine at  m/z  290.1369, C) cocaine at  m/z  304.1536 and D) cocaethylene at  m/z  318.1627.

Figure 2. MALDI-FTICR-MS imaging of cocaine user hair samples. A) Optical image of longitudinally sectioned hair samples following matrix application. B) MALDI-FTICRMS images showing the distribution of B) benzoylecognine at m/z 290.1369, C) cocaine at m/z 304.1536 and D) cocaethylene at m/z 318.1627.

 
 

MALDI-FTICR-MS Imaging of carbamazepine and metabolites in user hair samples

In this study, the use of MALDI-MS imaging to monitor the distribution of medications in patient hair samples was also investigated. Carbamazepine is an anti-seizure medication used in the management of epilepsy. Hair samples were obtained from a patient taking 800 mg of carbamazepine/day. The MALDI-FTICR-MS images show the distribution of carbamazepine at m/z 237.1022 (Figure 3B) and the major metabolite, carbamazepine-10, 11-epoxide at m/z 253.0971 (Figure 3C). The images shows consistent use of the drug which correlates well with a daily dose, indicating the patient is complying with their prescribed medication use. The length of the hair is 5 cm, which corresponds to 5 months of growth.

 
 
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Conclusions

The use HTX TM-Sprayer for the application of the matrix onto longitudinally sectioned hair samples has been demonstrated. Microscopy showed the matrix was homogenously distributed over the surface of a longitudinally sectioned hair sample. MALDI-FTICR-MS imaging showed the presence of cocaine and for the first time the anti-seizure medication carbamazepine in hair samples, which shows that MALDI-MS imaging could also be used to also monitor patient compliance to medication as well as forensic toxicology. We have also successfully used this method to look at the distribution of other compounds in the hair.

References

(1) Pragst F, Balikova MA. State of the art in hair analysis for detection of drug and alcohol abuse. Clin. Chim. Acta. 2006;370(1-2):17-49.

(2) Kintz P, Villain M, Cirimele V. Hair analysis for drug detection. Ther. Drug Monit.

2006;28(3):442-446.

(3) Porta T, Grivet C, Kraemer T, Varesio E, Hopfgartner G. Single hair cocaine consumption monitoring by mass spectrometric imaging. Anal. Chem. 2011;83(11):4266-4272.

(4) Miki A, Katagi M, Kamata T, et al. MALDI-TOF and MALDI-FTICR imaging mass spectrometry of

methamphetamine incorporated into hair. J. Mass Spectrom. 2011;46(4):411-416.

(5) Flinders B, Cuypers E, Zeijlemaker H, Tytgat J, Heeren RM. Preparation of longitudinal sections of hair samples for the analysis of cocaine by MALDIMS/ MS and TOF-SIMS imaging. Drug Test. Anal. 2015;7(10):859-865.

(6) Flinders B, Beasley E, Verlaan RM, et al. Optimization of Sample Preparation and Instrumental Parameters for the Rapid Analysis of Drugs of Abuse in Hair samples by MALDI-MS/MS Imaging. J. Am. Soc. Mass Spectrom. 2017;28(11):2462-2468.