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Despite the fact that strong routine separation methodologies can give reliable specificity and validity at usual working pharmaceutical concentrations, they may fail at very low concentration levels. This poses considerable challenges for researchers investigating product purity and therapeutic drug monitoring. Sensitivity enhancement procedures are thus required to maximize the performance of separation techniques. Solid-phase extraction/solid-phase enrichment (SPE/SPEn) and pre-, post-, and in-column derivatization, as well as the use of sensitive detection devices, are the simplest strategies for improving sensitivity of separation-based analytical techniques. Large-volume injection of samples with online SPE/SPEn coupled with separation techniques increased sensitivity and improved detection as well as quantification limits without affecting peak shape and system performance. Although the primary purpose of derivatization is to improve sensitivity and selectivity, greener derivatization is growing in popularity and should be considered in analytical chemistry. In general, two strategies are essential for accomplishing greener derivatization goals. The first is the search for and use of ecologically acceptable derivatizing reagents, solvents, and reaction conditions. The second is miniaturization and automation of analytical methods. This review discusses significant advances in separation-based analytical techniques, specifically enrichment approaches and detector signal improvement for pharmaceutical quantification in various matrices at very low concentration levels. As a result of improved analytical systems setup in drug assays, the possibility of high-throughput analyses was also highlighted.

A simple, selective, rapid, sensitive and less costly green automated solid phase extraction bio-analytical high-performance liquid chromatographic-based technique with fluorescence detection (Aut-SPE-BA-HPLC-FL) for the quantification of levofloxacin in human serum samples has been developed and validated. The serum samples were loaded into the chromatographic system without prior treatment and then injected into short (20 mm × 4.6 mm, 20 µm) protein-coated (PC) µBondapak CN (µBCN) silica pre-column (PC-µBCN-pre-column). Levofloxacin was retained and pre-concentrated on the head of the PC-µBCN-pre-column, while proteins and other polar components were eliminated using phosphate buffer saline (PBS), pH 7.4, as the first mobile phase in the extraction step. Levofloxacin is then transferred to the analytical column; ZORBAX Eclipse XDB-C18 (150 mm × 46 mm, 5 µm), through the aid of a column-switching valve technique, on-throughs the elution mode using the second mobile phase containing a methanol and phosphate buffer (0.05 M, pH 5) in a ratio of 70:30 (v/v). Levofloxacin signals were detected using a fluorescence detector operated at excitation/emission wavelengths of 295/500 nm. The proposed Aut-SPE-BA-HPLC-FL methodology showed linearity over a levofloxacin concentration range of 10–10,000 ng/mL (r2 = 0.9992), with good recoveries ranging from 87.12 to 97.55%. Because of the validation qualities in terms of linearity, recovery, precision, accuracy, selectivity and robustness, the Aut-SPE-BA-HPLC-FL method has been used in some clinical trials for therapeutic drug monitoring and the pharmacokinetic study of levofloxacin in human serum.

An automated solid phase extraction (SPE) protocol to determine carbamazepine in human serum has been developed and validated using a simple, rabid and sensitive liquid chromatography-based bio-analytical method. Extraction of carbamazepine was carried out using an on-line SPE tool of a short protein-coated (PC) ODS silica pre-column (PC-ODS-pre-column) and phosphate buffer saline (PBS) with a pH of 7.4 as an extraction solvent. There are two distinct chromatographic modes used by PC-ODS-pre-column. While carbamazepine trapping required reversed-phase liquid chromatography, proteins were extracted from serum samples using PBS by size-exclusion liquid chromatography. Then, carbamazepine was eluted from the PC-ODS-pre-column onto the quantification position using a mixture of methanol-distilled deionized water (50:50, v/v) as an eluent and ODS analytical column. At room temperature (22 ± 1 °C), carbamazepine was completely separated from the co-eluted matrix components and detected at 230 nm. Carbamazepine’s linearity was obtained at concentrations ranging from 50 to 10,000 ng/mL. With good accuracy and precision, carbamazepine recoveries in serum samples ranged from 86.14 to 97.82%. The extraction step was conducted using PBS as a safe and green extraction solvent, making this protocol both cost-effective and ecologically safe.

An in-capillary derivatization (in-CAP-D) integrated with field amplified sample stacking (FASS) has been developed for the determination of morphine (MOR) and its metabolite, morphine-6-glucuronide (MOR-6-G) in human serum using capillary zone electrophoresis (CZE) and fluorescence detection (in-Cap-D-FASS-CZE). Acetonitrile was employed in removing proteins and extracting MOR and MOR-6-G into the clear supernatant containing codeine (COD) as an internal standard (IS). The derivatization was achieved in an in-capillary mode by introducing the acetonitrile-treated samples into a running electrolyte containing an oxidizing agent of potassium ferricyanide, whereas MOR, MOR-6-G and COD were oxidized into dimer derivatives with highly fluorescent intensity. The effectiveness and sensitivity of the in-Cap-D-FASS-CZE method were affected by many parameters, and the following conditions were found to be optimal: 70 m Mdisodium tetraboratedecahydrate (pH, 10.5), 0.30 mM ferrricyanide and a separation voltage of 10 kV. In order to perform the FASS, samples were electrokinetically injected for 20 s at 20 kV into the capillary that was pre-field with a 4 s water plug. Analysis was performed at ambient temperature (22 ± 1 °C). The method’s validation revealed good linearity with respect to peak area ratios of MOR and MOR-6-G with the IS and the corresponding concentrations over the ranges of 1–2000 and 1.2 to 2000 ng/mL, respectively. Following one oral dose of controlled-release MOR sulphate tablet, the validated in-Cap-D-FASS-CZE method successfully enabled the determination of MOR and MOR-6-G in clinical serum samples.