Improved and validated method for the determination of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC in serum,and in human liver microsomal preparations using gas chromatography-mass spectrometry

A validated method for the quantification of Delta(9)-tetrahydrocannabinol (THC) and its main metabolites 11-hydroxy-tetrahydrocannabinol (OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) in serum is presented. The substances were isolated by solid-phase extraction, derivatised by methylation, and analysed by means of GC-MS in the selected ion monitoring mode. Quantitation was achieved by the addition of deuterated analogues as internal standards. The method was linear up to 10 ng/ml for THC and OH-THC, and up to 50 ng/ml for THC-COOH. The limits of quantification were 0.62 ng/ml for THC, 0.68 ng/ml for OH-THC and 3.35 ng/ml for THC-COOH. The limits of detection for the least intensive ions were 0.52 ng/ml for THC, 0.49 ng/ml for OH-THC and 0.65 ng/ml for THC-COOH. The method was validated according to the requirements of the Journal of Chromatography B. The method has been routinely used on samples from drivers suspected of "driving under the influence". In addition to the forensic application, a cross-validation was carried out by applying the method developed for serum to human liver microsomal preparation samples.     

High-throughput, semi-automated determination of a cyclooxygenase II inhibitor in human plasma and urine using solid-phase extraction in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6′-methyl-[2,3′]bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard (II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C₈ SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C₈/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP^® guard column (20×4.6 mm) connected to a Prism-RP^® analytical column (150×4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH=4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (λ_ex=260 nm, λ_em=375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5–500 ng/ml for human plasma and human urine yielded a linear response (R²>0.99) when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.     

Simple liquid chromatography method for the rapid simultaneous determination of prednisolone and cortisol in plasma and urine using hydrophilic lipophilic balanced solid phase extraction cartridges

This article describes the development and validation of a simple solid phase extraction (SPE) and HPLC method for the extraction and the specific determination of prednisolone and hydrocortisone (cortisol) in both plasma and urine using one washing step with Oasis hydrophilic lipophilic balanced (HLB) cartridges (1 ml/30 mg, 30 microm). Recoveries of prednisolone and cortisol from plasma and urine exceeded 82%. The limit of quantification (LOQ) in plasma and urine was 9.9 and 6.7 ng/ml for cortisol, respectively, and 11.6 and 8.0 ng/ml for prednisolone, respectively. The intraday and interday precision (measured by CV%) for both prednisolone and cortisol in both plasma and urine was always less than 7%. The accuracy (measured by relative error %) for both prednisolone and cortisol in both plasma and urine was always less than 8%. The advantages of the developed method are the use of a one step washing SPE utilising HLB cartridges which do not suffer the drying out problems of conventional SPE cartridges and the time saving when compared with solvent extraction (SE), in addition to the simultaneous determination of prednisolone and cortisol in both plasma and urine.     

Development of a LC/MS/MS method for the analysis of cannabinoids in human EDTA-plasma and urine after small doses of Cannabis sativa extracts

A novel high-performance liquid chromatographic separation method with tandem-mass spectrometry detection was developed for the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and its major metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) as well as the components cannabidiol (CBD) and cannabinol (CBN) in human EDTA-plasma and urine. Run time was 25 min. Lower limit of quantification was 0.2 ng/ml. The coefficients of variation of all inter- and intra-assay determinations were between 1.3 and 15.5%. The method was successfully applied to the determination of cannabinoids in human plasma and human urine after administration of Delta(9)-tetrahydrocannabinol or Cannabis sativa extracts.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

High-performance liquid chromatographic procedures for the quantitative determination of paclitaxel (Taxol) in human urine

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the quantitative determination of paclitaxel in human urine. A comparison is made between solid-phase extraction (SPE) and liquid-liquid extraction (LLE) as sample pretreatment. The HPLC system consists of an APEX octyl analytical column and acetonitrile-methanol-0.2 μM ammonium acetate buffer pH 5 (4:1:5, v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The SPE procedure involves extraction on Cyano Bond Elut columns. n-Butylchloride is the organic extraction fluid used for the LLE. The recoveries of paclitaxel in human urine are 79 and 75% for SPE and LLE, respectively. The accuracy for the LLE and SPE sample pretreatment procedures is 100.4 and 104.9%, respectively, at a 5 μg/ml drug concentration. The lower limit of quantitation is 0.01 μg/ml for SPE and 0.25 μg/ml for LLE. Stability data of paclitaxel in human urine are also presented.     

Simultaneous determination of blood concentrations of methohexital and its hydroxy metabolite by gas chromatography and identification of 4′-hydroxymethohexital by combined gas—liquid chromatography—mass spectrometry

A simple, sensitive and selective method is described for the simultaneous determination of low concentrations (less than 50 ng/ml) of underivatized methohexital and its hydroxy metabolite in small (0.1 ml) samples of human and rat plasma or whole blood by gas chromatography with nitrogen-selective detection.Moreover, the main metabolite in rat and man was identified as 4′-hydroxymethohexital by comparison of chromatograms from gas—liquid chromatography (GLC) with data obtained from GLC—mass spectrometry and ¹H-nuclear magnetic resonance spectrometry of this metabolite, produced both by incubating methohexital with isolated rat liver microsomes and by isolating this metabolite from rat urine.     

Solid-phase extraction–high-perfomance liquid chromatography determination of verapamil and norverapamil enantiomers in urine

A simple, rapid, sensitive and selective method has been developed for the stereospecific determination of verapamil and its metabolite, norverapamil in urine. For sample preparation we utilized a membrane-based solid-phase extraction (SPE) disk consisting of a thin, particle-loaded membrane inserted in a plastic syringe-like barrel. The particles, which may be C₈ or C₁₈ bonded phase (C₈ in this work), are embedded within a matrix of PTFE (Teflon) fibrils. Overall analyte recoveries were above 85%, even at low concentration of 3.0 ng/ml with reproducibilities (C.V. values) below 13.1%. This method of extraction has the advantage of speed and considerable reduction in solvent volumes compared to conventional SPE and solvent extraction. The separation of all the enantiomers was achieved using a single chiral stationary phase column, the cellulose-based reversed-phase, Chiralcel OD-R. Analyte concentrations of less than 3.0 ng/ml could be quantitated with C.V. values below 14%. Calibration curves were linear in the range 2.5–300 ng/ml. Intra-day and inter-day reproducibilities were 10.5–14.2% at 3 ng/ml, 4.8–9.3% at 138.5 ng/ml and 7.8–10.1% at 280 ng/ml level, respectively, for all the enantiomers.     

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 μm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min⁻¹ the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml⁻¹ for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml⁻¹ for plasma samples, and <4% in the concentration range of 40-400 ng ml⁻¹ for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 μm C₁₈ column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min⁻¹ the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml⁻¹ for apomorphine and 2.5 ng ml⁻¹ for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml⁻¹ for plasma samples and 7% in the concentration range of 50-500 ng ml⁻¹ for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with β-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 μg kg⁻¹ for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (Cipralan ^TM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile---phosphate buffer (0.015 mol/1, pH 6.0) (80:20). A 10-μ ion-exchange (sulfonate) column was used with acetonitrile—phosphate buffer (0.015 mol/1, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard.The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10–1000 ng/ml and 50–5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Rapid reversed-phase high-performance liquid chromatographic method for the assay of urinary 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid and confirmation of use of cannabis derivatives

The main active cannabis (Marijuana and hashish) derivative Δ⁹-tetrahydrocannabinol is, in vivo, transformed and excreted mainly as 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide. The method presented here allows the confirmation of the presence of THC-COOH by means of a basic hydrolysis, solid-phase extraction clean-up on reversed-phase (RP) disposable cartridges followed by analysis on a C₈ RP column and UV detection; the mobile phase used was a 55% acetonitrile solution in acid phosphate buffer. Over 600 samples both from drug addicts in therapeutic communities and subjects who were not on any drugs therapy were analysed. This method was precise with a linearity range from 10 to more than 500 ng/ml [the lower limit proposed by the National Institute on Drug Abuse (NIDA) for cannabinoid confirmation method is 15 ng/ml]. The sample preparation is simple and fast, allowing the analysis of large numbers of samples. Perfect correlation was observed between data from the HPLC method and a fluorescence polarization immunoassay screening method. The THC-COOH metabolite was found to constitute 30% of all the cannabinoids excreted in urine of abusers.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

Determination of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine using high-performance liquid chromatography and electrospray ionization mass spectrometry

High-performance liquid chromatography with electrospray ionization mass spectrometry was used to determine 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine. After basic hydrolysis of conjugates, the compound was extracted using SPEC-PLUS-3ML-C₁₈ solid-phase extraction columns. A deuterium labelled internal standard (d₃-THC-COOH) was added prior to hydrolysis. Separation was performed on a reversed-phase Zorbax Eclipse XDB-C₈ analytical column (150×3.0 mm I.D.) using a gradient program from 60 to 80% acetonitrile (4 mM formic acid) at a flow-rate of 0.5 ml/min. The compounds were detected by single ion monitoring of m/z 345 and m/z 348 for the protonated molecules [THC-COOH+H]⁺ and [d₃-THC-COOH+H]⁺, respectively. The precision and accuracy were tested on spiked urine samples in the range 2.5–125 ng/ml. The mean recovery was 95% (n=58), coefficients of variations were 2.2–4.3% and the limit of detection 2 ng/ml. Diagnostic qualifying ions of THC-COOH (m/z 327 and m/z 299) and d₃-THC-COOH (m/z 330) were generated using up-front collision-induced dissociation. The relative ion intensities in clinical samples (n=21) were within ±20% deviation compared with standards. Using this tolerance and the presence of the ions m/z 327 and m/z 299 at the correct retention times as the acceptance criteria for identification of THC-COOH positive samples, the limit of detection was 15 ng/ml. The LC–MS method complies with the current recommendations on drugs of abuse testing, in which mass spectrometric detection is emphasized.     

Determination of stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume

Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025–25 and 2–150 μg/ml in plasma and urine, respectively. An aliquot of 200 μl of plasma was extracted with solid-phase extraction using Oasis^® cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 μl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.     

Achiral and chiral high-performance liquid chromatography of verapamil and its metabolites in serum samples

Rapid and simple achiral and chiral HPLC assays have been developed for the determination of verapamil and its metabolites in serum samples. Two achiral reversed-phase columns, Hisep C₁₈ (150×4.6 mm) and NovaPak C₁₈ (150×3.9 mm) were used for the simultaneous separation of all analyzed compounds. An α₁-AGP column (100×4.0 mm) was recommended for successful chiral separations of verapamil and its seven metabolites. All analyses were realised with fluorescence detection at λ_ex=276 nm and λ_em=310 nm. Limits of quantitation were in the range 1.0 to 5 ng/ml for all compounds. Both off-line SPE (SepPak C₁₈ cartridges) and the on-line SPE with a semipermeable surface SDS C₈ pre-column, (10×4.6 mm) were used for the clean-up and sample preconcentration. Extraction recoveries for all analyzed compounds were 87.7±5.8 to 92.7±4.0% for off-line SPE and 94.3±4.2 to 98.2±5.1% for on-line SPE. The complete assay could be applied for achiral and chiral monitoring verapamil and all its metabolites in serum samples.     

Development and validation of a method for the quantitation of Delta9tetrahydrocannabinol in oral fluid by liquid chromatography electrospray-mass-spectrometry

Analysis of Delta(9)tetrahydrocannabinol (Delta(9)THC) and its metabolites in biological samples is of great relevance for forensic purposes. In the case of oral fluid (OF), the analysis should determine Delta(9)THC, whereas in urine, it detects the inactive metabolite tetrahydrocannabinol carboxylic acid (THC-COOH). Most laboratories analyze Delta(9)THC in such samples using GC-MS methods, but these procedures are time-consuming and involve unavoidable previous extraction and derivatization. No data is yet available on the application of liquid chromatography-mass-spectrometry to detect Delta(9)THC in oral fluid. We report a validation method in which the Delta(9)THC is isolated from oral fluid by a simple liquid-liquid extraction with hexane and subsequently analyzed by liquid chromatography-mass-spectrometry. The method here reported for the determination of Delta(9)THC in oral fluid only requires 200 microl of sample and achieves limits of detection of 2 ng/ml, and has been used to analyze oral fluid samples collected from current drug users.     

Simultaneous determination of buprenorphine, norbuprenorphine, and buprenorphine–glucuronide in plasma by liquid chromatography–tandem mass spectrometry

For the first time, an LC–MS–MS method has been developed for the simultaneous analysis of buprenorphine (BUP), norbuprenorphine (NBUP), and buprenorphine–glucuronide (BUPG) in plasma. Analytes were isolated from plasma by C₁₈ SPE and separated by gradient RP-LC. Electrospray ionization and MS–MS analyses were carried out using a PE-Sciex API-3000 tandem mass spectrometer. The m/z 644→m/z 468 transition was monitored for BUPG, whereas for BUP, BUP-d₄, NBUP, and NBUP-d₃ it was necessary to monitor the surviving parent ions in order to achieve the required sensitivity. The method exhibited good linearity from 0.1 to 50 ng/ml (r²≥0.998). Extraction recovery was higher than 77% for BUPG and higher than 88% for both BUP and NBUP. The LOQ was established at 0.1 ng/ml for the three analytes. The method was validated on plasma samples collected in a controlled intravenous and sublingual buprenorphine administration study. Norbuprenorphine–glucuronide was also tentatively detected in plasma by monitoring the m/z 590→m/z 414 transition.     

An improved on-line solid phase extraction coupled HPLC–MS/MS system for quantification of Sifuvirtide in human plasma

An improved liquid chromatographic method with on-line solid phase extraction (SPE) and tandem mass spectrometric detection was optimised for quantification of the anti-HIV peptide Sifuvirtide in human plasma. The SPE sorbents, loading buffer composition and other aspects of the on-line SPE column were investigated in detail for efficiently extracting the interesting peptides and simultaneously discarding the large amount of proteins. The gradient elution program was optimised on the analysis column to decrease the matrix effect and obtain excellent selectivity. The multiple charge ion at m/z 946.4 of Sifuvirtide was quantified by a linear ion trap mass spectrometer, operating in the positive mode, and selective reaction monitoring (SRM) acquisition. Method validation results demonstrated that the linear calibration curve covered a range of 6.1–6250 ng/mL, and the correlation coefficients (r²) were above 0.992. The lower limit of detection (LLOD) with a signal-to-noise (S/N) ratio higher than 10 was 6.1 ng/mL. The accuracy ranged from −7.6 to 10.6%, and the intra- and inter-batch precisions were less than 8.7% and 5.5%, respectively. Finally, more than nine hundred of samples from a clinical trial was completely analyzed using this on-line SPE coupled HPLC–MS/MS system in one single week, due to the rapid run-time of individual sample (6.5 min).