In the pharmaceutical industry, liquid chromatography–tandem mass spectrometry (LC–MS-MS) is already an established method for quality control and quantification of drugs in different matrices. Additionally, this hyphenated analytical tool is becoming more and more important in clinical chemical analysis (that is, in therapeutic drug monitoring).

LC–MS-MS is a very powerful analytical technique because it combines the separation power of LC with the sensitivity and selectivity of MS. However, LC–MS-MS possesses two major drawbacks when analyzing drugs in biological fluids. The first, associated with the mass spectrometer, is that the electrospray source is very susceptible to matrix-related ion-suppression effects. The second drawback concerns the LC. Because of irreversible adsorption and precipitation effects caused by high molecular weight sample components (for example, proteins) complex biofluids cannot be injected and analyzed directly.

Consequently, the analysis of complex biological fluids generally requires pretreatment steps aimed at the removal of unwanted matrix constituents from the sample. In bioanalytical LC, solid-phase extraction (SPE) is the predominant clean-up technique. However, the high matrix load of complex biofluids affects the efficiency of this extraction technique and gives rise to co-elution of interfering substances. This is particularly true for proteins, because many commercially available SPE sorbents are not biocompatible and cause nonspecific adsorption and/or precipitation of proteins. With on-line SPE, this in turn causes a clogging of the SPE column and shortens its lifetime dramatically. As a result, most sample clean-up procedures include a protein precipitation step in order to prevent these effects. However, protein precipitation cannot be automated easily and, in fact, Polson and colleagues (1) showed that protein precipitation is not efficient enough to remove all substances that cause ion suppression. In addition, protein-bound analytes are often co-precipitated. Thus, optimization of sample pretreatment should be directed toward improved efficiency–selectivity and automation. We have accomplished this with the development of a multidimensional SPE platform for extractive on-line clean-up and subsequent LC–MS-MS analysis of drugs with basic properties and low polarity in raw biofluids (Figure 1).

Figure 1: Schematic of the MD-SPE operational procedure. Analyte 5 squares, high molecular weight matrix component 5 triangles, low molecular weight matrix component 5 circles.

ExperimentalInstrumental set-up formulations: Figure 2 illustrates the multidimensional on-line SPE–LC platform. The system consists of three conventional HPLC pumps, two six-port switching valves controlled by an HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland), a diode array detector (Merck KGaA, Darmstadt, Germany), a restricted access material (RAM) SPE column (5 × 4 mm i.d.) packed with LiChrospher^® ADS RP18 material (Merck KGaA, Darmstadt, Germany), a mixed mode polymer (MMP) SPE column (20 × 1 mm i.d., research prototype provided by Waters Corporation, Milford, Massachusetts), and an analytical column (Zorbax RX-C18, 3.5 µm, 75 × 4.6 mm i.d., Agilent Technologies, Waldbronn, Germany).

Figure 2: Schematic of the MD-SPE-LC system. 1 5 pumps, 2 5 autosampler, 3 5 waste, 4 5 RAM column, 5 5 MMP column, 6 5 analytical column, 7 5 detector, V1 and V2 5 valves.