Development, validation and application of an HPLC–MS/MS method for the determination of fentanyl and nor-fentanyl in human plasma and saliva

Abstract

Monitoring fentanyl concentration in saliva and plasma may be useful in pharmacokinetic/pharmacodynamic studies. Salivettes(®) have been used widely for collecting saliva samples. However due to its lipophilicity, fentanyl adsorbs to the cotton dental bud (CDB) used in this device. Furthermore, due to dry mouth being a common adverse effect seen in patients treated with opioids, obtaining enough saliva for analysis is often a challenge. Hence, a simple simultaneous method to quantify fentanyl and its metabolite in both human plasma and saliva was developed and validated. A novel extraction method was also developed and validated to recover fentanyl in saliva directly from the CDB. This extraction method utilises acetonitrile to recover the fentanyl directly from the CDB rather than recovery by centrifugation, which is not always possible. Reverse phase chromatographic separation was performed on a Shimadzu LC 20A HPLC system using gradient elution. The electrospray ion source (ESI) was operated in positive ion mode using an Applied Biosystems API 3200 LC/MS/MS as detector. Deuterated fentanyl (D5) and nor-fentanyl (D5) were used as internal standards (IS). The retention times for fentanyl and nor-fentanyl were 3.70 min and 3.20 min respectively. The lower limit of quantitation (LLOQ) was determined to be 0.030 μg/L in plasma and 0.045 in saliva for fentanyl and nor-fentanyl. Acceptable linearity for fentanyl and nor-fentanyl in both plasma and saliva was demonstrated from 0.02 to 10 μg/L (R(2) 0.9988-0.9994). Accuracy for fentanyl and nor-fentanyl in both plasma and saliva samples was between 96% and 108%. Total imprecision expressed as the co-efficient of variation was between 1.0 and 15.5% for both analytes in both matrices. The validated method was applied successfully in 11 paired plasma and saliva samples obtained from patients with cancer pain receiving transdermal fentanyl (Duragesic(®)) at doses from 25 μg to 100 μg.

Effectiveness of saliva and fingerprints as alternative specimens to urine and blood in forensic drug testing

“…In addition, only dilution and deproteinization using water and MeCN were used to simplify the pretreatment before the analysis and a small volume of the specimen (5 μL) was diluted with a large volume of the solvent (550 times) to reduce the matrix effects. Generally, a larger specimen volume (>100 μL) is used, and more complicated procedures, such as extraction, evaporation, and filtration, are required to remove matrix interference . Although the sample preparation for the urine, blood, and saliva samples used in this study was simple and rapid when compared to common methods, it still required approximately 15 min for each sample.…”

“…Generally, a larger specimen volume (>100 μL) is used, and more complicated procedures, such as extraction, evaporation, and filtration, are required to remove matrix interference. [4][5][6][7][8][13][14][15] Although the sample preparation for the urine, blood, and saliva samples used in this study was simple and rapid when compared to common methods, it still required approximately 15 min for each sample. However, the sample preparation for the fingerprint samples was simpler and faster (only requiring addition of solvent, shaking, and centrifugation) because fingerprints had less matrix components than urine, blood, or saliva.…”

“…There are many reports regarding drug analysis in saliva, and the relationship of drug concentrations between blood and saliva has been independently investigated for various compounds . Although drug detection in fingerprints has been reported recently, there are few reports regarding the detection of administered drugs from fingertip secretions immediately after washing to remove external contaminants from contact with the subject's own body or other objects …”

“…[1][2][3] There are many reports regarding drug analysis in saliva, and the relationship of drug concentrations between blood and saliva has been independently investigated for various compounds. [4][5][6][7][8] Although drug detection in fingerprints has been reported recently, [9,10] there are few reports regarding the detection of administered drugs from fingertip secretions immediately after washing to remove external contaminants from contact with the subject's own body or other objects. [11,12] Herein, 14 analytes (the administered drugs and their main metabolites) were monitored in urine, blood, saliva, and fingerprints for approximately 4 weeks after 4 pharmaceutical products were administered.…”

Confirmation and Analysis of Fentanyl and Its Analogues Without the Standards

“…At the beginning of 2014, the availability of fentanyl in the United States had started to increase exponentially [4], with crime laboratories reporting more than 4,500 fentanyl cases in 2014 and over 14,400 Numerous analysis methods for fentanyl and its analogues have been published in the scientific literatures. Analysis technique such as liquid chromatography (HPLC) [5][6][7][8][9], gas chromatography/mass spectrometry (GC/MS) [10][11][12][13][14][15][16][17][18], liquid chromatography/high resolution mass spectrometry (LC-HRMS) [19][20], liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) [21][22][23][24][25][26][27][28][29][30] has been reported to apply in the precision determination and quantification of fentanyl and its analogues. GC/MS analysis method has been considered a gold standard for decades due to its combination of good selectivity and high specificity.…”