Fast Analysis of "Dirty" Samples with Monolithic Silica LC and Silica Gel TLC Stationary Phases

Jul 01, 2014

This article describes the development of fast and straightforward processes of sample preparation, separation, and detection for the analysis of various food and pharmaceutical samples with a high matrix load. The use of stationary phases such as monolithic silica columns or silica gel thin-layer chromatography (TLC) plates allows for no sample preparation or very simple sample preparation protocols. After the chromatographic separation, samples were analyzed using UV–vis or mass spectrometry (MS) detection. In the case of TLC, MS analysis was performed by applying a combination of a TLC–MS interface and a compact mass spectrometer. The developed setups enabled the identification of colorants, isoflavones, and caffeine in drinks as well as beta blockers and paracetamol in human urine and drug formulations.

Food and pharmaceutical analyses typically involve the examination of "dirty" samples that are contaminated with particulate matter and also may contain high amounts of dissolved substances. The analysis of food is required to confirm a specified composition, to meet regulatory aspects, and to protect products against fraud. In the pharmaceutical environment the needs are similar; in addition, patient samples must be analyzed for abnormalities, drug molecules, or metabolites (1). In food analyses, the dissolved substances can be carbohydrates, proteins, lipids, or inorganic salts. In the pharma sector, bodily fluids such as blood (whole blood, serum, and plasma) or urine contain proteins, urea, inorganic salts, and organic acids. The main matrix compounds (excipients) of drug formulations are partly soluble additives and binding materials (for example, starch, lactose, or microcrystalline cellulose in pills or glycerides in suppositories) (2).

Sample analysis is a three-step process of sample preparation, separation via chromatography as the most widely used technique, and analyte detection with ultraviolet–visible (UV–vis) spectroscopy or mass spectrometry (MS). In general, sample handling should be minimized to allow for cost-effective investigations. Separation using robust liquid chromatography (LC) columns with a long lifetime or high-performance thin layer chromatography (HPTLC) plates displaying a high matrix tolerance is desirable.

The sample preparation can be as simple as a dissolution step followed by precipitation, filtration, or a multistep solid-phase extraction (SPE) procedure. The sample preparation procedures strongly correlate to the subsequent separation experiment. Particle-packed LC columns often lack robustness and tend to clog, especially when the particle diameter is less than 2 µm; as a result, tedious and time-consuming sample preparation procedures are necessary. In contrast, monolithic silica stationary phases used with standard HPLC equipment are extremely robust and clogging is minimized because of their fritless design and specific pore structure. TLC is a third option for the separation of samples with very high matrix load, and it requires no sample preparation. It is an effective, low-cost, and rather simple method used for the analysis of organic compounds in food, pharmaceutical, and environmental samples. Next to the high matrix tolerance of TLC, another huge advantage is the possibility of performing many chromatographic runs in parallel on one plate (3). Today, all steps from sample application to detection have been automated, which makes reliable quantitative and qualitative analyses possible. Recent developments allow for off-line TLC-MS, adding additional value by generating data about molecular masses and detailed structural parameters of target molecules (4). In addition, MS is by far more sensitive in most applications than widely used detectors such as UV–vis or fluorescence detectors.

Hence, extensive sample preparation procedures can be avoided in most cases when using either monolithic silica columns or TLC plates as stationary phases.

Most analytical laboratories use UV–vis or MS detectors in the final step of sample analysis. The former is a quite unspecific system, but it can handle a high matrix load very well and maintenance intervals are more or less independent of the nature of the analyzed sample. On the other hand, a mass spectrometer delivers precise and specific data about the molecular weight and molecular structure of analytes. But the robustness of the detector is comparably lower, and contamination with matrix components deteriorates reproducibility and accuracy (so-called matrix effects caused by ion suppression or ion enhancement — for example, as might be encountered in quantification) and increases maintenance costs.

As a consequence, the choice of both the detector and the chromatographic column or plate has a direct impact on the efforts to be made during sample preparation.

Here, we present a set of application examples describing the analysis of different food and pharmaceutical samples on a monolithic silica column and with UV–vis or MS detection. The sample preparation protocols were kept as short as possible to meet the requirements of both the stationary phase and the detector, and fast chromatographic separations were achieved via short gradient runs.

In a second set of experiments, HPTLC–MS was used for the determination of analytes in complex sample matrices without applying any tedious classical sample preparation steps. Two methods were developed for the parallel analysis and quantification of caffeine and paracetamol using MS-grade HPTLC silica gel plates and an elution-based TLC–MS interface coupled with a compact mass spectrometer. To enable high sensitivity and low background signals, special MS-grade silica gel HPTLC plates were applied in this setup.


Materials and Methods

High Performance Liquid Chromatography

The high performance liquid chromatography (HPLC) system used was a Dionex Ultimate 3000 (Thermo Scientific Dionex Corporation) equipped with a UV–vis detector (detection wavelength 500 nm). The data acquisition was performed with Chromeleon software.

Separation was performed on 50 mm × 2 mm Chromolith FastGradient RP-18 endcapped analytical monolithic silica columns and 5 mm × 2 mm Chromolith RP-18 endcapped guard cartridges (both Merck Millipore).

A Bruker Esquire 6000 mass spectrometer with an ion trap and an on-line electrospray ionization (ESI) source operated in positive mode was used in the m/z 100–600 scan range (depending on the sample).


Images of HPTLC plates were taken under UV irradiation (254 nm). The direct extraction of HPTLC plates was performed using a TLC–MS interface (CAMAG). The interface was directly connected to an Expression CMS mass spectrometer (Advion) operated in positive ESI mode (m/z range 100–300). TLC plates (20 cm × 10 cm HPTLC silica gel 60 F254 MS-grade glass plate) were extracted at 0.1 mL/min with 95:5 (v/v) acetonitrile–water plus 0.1% formic acid. All solvents, reagents, and TLC plates were purchased from Merck Millipore.