An Online post-column solvent-assisted ionization (OPSAI) method was developed for enhancing the ionization of the beta-blocker propranolol utilizing normal phase LC-MS/MS. Solvent-assisted electrospray ionization (SAESI) was studied by the introduction of the assistant solvents A: 0.5% Formic acid in Isopropanolol, B: 0.5% Formic acid in lsopropanolol-Water (1:1), and C: 0.5% Formic acid in water into the electrospray ionization chamber using a spray needle. Analyte molecules can be directly ionized by the aid of the assistant solvent spray. Both methods were applied to the chiral separation of propranolol enantiomers using normal phase analysis on cellulose-based chiral column. Interestingly, both methods are easy to handle and offer a wide range of assistant solvents that can be used in order to gain the optimum ionization of the analyte molecules. The both methods considerably improved the analyte signal and the peak area greatly increased. The propranolol average signal-to-noise (S/N) ratio was enhanced from 26 +/- 1 and 42 +/- 1 to 2341 +/- 61 and 1725 +/- 29 for R-propranolol and S-propranolol, respectively, when the post-column solvent method (OPSAI) was used with isopropanol-assistant solvent (A). While in case of solvent-assisted electrospray ionization method (SAESI) signal was enhanced from 26 +/- 1 and 42 +/- 1 to 2223 +/- 72 and 2155 +/- 58 for R-propranolol and S-propranolol, respectively, with water as an assistant solvent. The limit of detection was 10 ng/mL and the method was linear in the range 50-2000 ng/mL. The NPLC-MS method was applied for the determination of propranolol enantiomers in human plasma after microextraction by packed C18 sorbent.

A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography-electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose-SB column (150x4.6mm, 5m) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane-isopropanol (80:20, v/v) with a flow rate of 0.8mL/min. A post-column solvent-assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2mL/min. The total chromatographic run time was 10min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.0872.09 for metoprolol and m/z 303.3154.3 for IS. The linearity range was 2.5-500ng/mL for both R- and S-metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5ng/mL respectively, in both matrices (plasma and saliva). The intra- and inter-day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories.

Being performance enhancing hormones, endogenous anabolic androgenic steroids (EAAS) are banned from most competitive sports by the World Anti-doping Agency (WADA).. In anti-doping control laboratories, routine assays are mainly performed on urine samples of athletes in and out of competitions. Serum constitutes a promising alternative to urine as it is less subjected to manipulation or contamination issues that may influence the method sensitivity. The simultaneous determination of EAAS and their conjugated metabolites is very challenging due to their contradicting chemical behaviors at the ionization interface of the mass spectrometer. This may prejudice their detection or limit the method sensitivity. Herein, we have addressed these challenges and developed a new method for the simultaneous determination of unconjugated, sulphate and glucuronide EAAS (Androsterone, Etiocholanolone, testosterone, epitestosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione and 17a-hydroxyprogesterone) in human serum using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The use of mass spectrometric detection in full scan mode facilitated the study of the most versatile adducts for detection and quantitation. Using solid phase extraction as sample preparation protocol prior to analysis, the method limits of quantitation ranged from 0.006 to 7.904 ng/mL and the recoveries ranged from 70.2 to 96.5 %. The method calibration was performed in untreated serum representing realistic matrix composition with correlation coeffecients ranged from 0.9859 to 0.9988. Finally, the serum- levels of the investigated steroids were determined in 4 male and 1 female human subject to provide estimates of baseline levels based on individual values.

Detection of low doses of recombinant growth hormone is a challenge in anti-doping testing. Future testing leads toward longitudinal monitoring of IGF-I and P-III-NP in an endocrine module. Additional biomarkers, for example vitamin D binding protein, alpha-HS-glycoprotein, fibronectin 1 and decoin have been identified in different omics studies. Here we have longitudinally studied the usefulness of these putative biomarkers in relation to two weeks administration of low doses of recombinant growth hormone in healthy male volunteers. Moreover, we studied the hematological parameters included in the athlete biological passport as well as serum concentration of dihydrotestosterone-sulfate. Alpha-HS-glycoprotein decreased 25 % up to three weeks after treatment period (p˂0.05), whereas fibronectin increased 20 % during the treatment (p˂0.05). The addition of these two biomarkers increased the likelihood to detect recombinant growth hormone intake based on individual calculated thresholds in some of the participants as compared to the GH2000 score. A minor 10 % increase (p=0.003) in reticulocytes percentage and OFF-score was observed, but the changes did not lead to any atypical profile based on individual passport thresholds. Vitamin D binding protein, decoin and dihydrotestosterone-sulfate were not affected by recombinant growth hormone. Dihydrotestosterone-sulfate was negatively correlated with the IGF-I biomarker at baseline (R=-0.50, p=0.003) and post dose (R=-0.59, p=0.01). In conclusion we have verified Alpha-HS-glycoprotein and fibronectin 1 as promising future biomarkers for detecting low doses of recombinant growth hormone and further studies of these proteins are encouraged.