Toxicological screening of human plasma by on‐line SPE‐HPLC‐DAD: identification and quantification of acidic and neutral drugs

Abstract

A multi-analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on-line solid-phase extraction (SPE)-HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro-neutral, one strong anion-exchange, one weak cation-exchange) for on-line extraction, five HPLC-columns [one C18 (GeminiNX), two phenyl-hexyl (Gemini C6 -Phenyl, Kinetex Phenyl-Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre-treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion-exchanger SPE cartridge (StrataX-A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C6 -Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile-water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter-/intra-day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from -14 to 10%. A computer-assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi-drug intoxications are presented.

Drug stability in forensic toxicology

“…Pentobarbital and phenobarbital were deemed stable in human plasma samples stored at −15°C for 3 months (Mut et al, 2016). Phenobarbital was also deemed stable in fortified human plasma stored at −30°C for 30 days and stored at −80°C for 100 days (Dalmora et al, 2010; Serralheiro et al, 2013).…”

Development of a simple HPLC‐DAD multi‐analyte procedure and its application in cases evaluated by the Poison Control Center of São Paulo, Brazil

“…Alternatively, blood samples can provide a possible correlation between compound concentration and toxic effects (Li, Shen, Jiang, Huang, & Zhuo, ; Maurer, , , ; Montenarh et al, ; Montenarh, Hopf, Maurer, Schmidt, & Ewald, ; Pizzolato, Alda, & Barceló, ; Pragst, ; Verstraet & Peat, ; Vincenti et al, ). The most commonly used techniques for extraction or pre‐treatment of samples for chromatographic analysis are liquid–liquid extraction (LLE) (Meyer et al, ; Montenarh et al, ; Staeheli, Poetzsch, Kraemer, & Steuer, ), solid phase extraction (SPE) (Dalsgaard et al, ; Mut, Grobosch, Binscheck‐Domaß, & Frenzel, ; Mut, Grobosch, Binscheck‐Domaß, & Frenzel, ; Steuer, Forss, Dally, & Kraemer, ; Tang, Ching, Lee, Lam, & Mak, ), liquid‐phase microextraction (LPME) (Meng, Zhang, Meng, Zhu, & Zheng, ; Nuhu, Basheer, & Saad, ) and solid‐phase microextraction (SPME) (Aleksa et al, ; Gentili, Mortali, Mastrobattista, Berretta, & Zaami, ; Olszowy et al, ). However, considering that nowadays simpler and cost‐effective sample preparation techniques are preferred in bio‐analytical methods, the LLE has attracted attention for toxicological analysis (Meyer, Weber, & Maurer, ; Vosough, Ghafghazi, & Sabetkasaei, ).…”

“…Despite the existence of a GC–MS library, its application is limited due to its incompatibility with hydrophilic, thermolabile and non‐volatile analytes (Liu, Liu, Lin, & Ho, ; Peters & Remane, . Additionally, the described chromatographic procedures involve the use of high‐performance liquid chromatography (HPLC) with a DAD detector (Moreno, Navas, & Asuero, ; Mut et al, , ; Petruczynik et al, ; Takitane et al, ) coupled to a mass spectrometer (Arora et al, ; Montenarh et al, , ; Tang et al, ). HPLC detection has proven to be a useful analytical tool that provides a satisfactory detection, identification and quantification of substances for screening routine analyses (Petruczynik et al, ).…”