Application of HPLC Technology in Drug Detection

What is High-Performance Liquid Chromatography (HPLC) and How Does It Work?

High performance liquid chromatography (HPLC) is also called "high-speed liquid chromatography", "high-resolution liquid chromatography", "modern column chromatography" and so on. High performance liquid chromatography is an important branch of chromatography. It uses liquid as the mobile phase, and uses a high-pressure injection system to pump single solvents with different polarities or solvents and buffers with different mixing ratios into the column containing the stationary phase. After the components in the column are separated, they enter the detector for detection, so as to realize the analysis of the sample. HPLC technology has the following main advantages: the application of extremely fine particles (generally less than 10 μm), regular and uniform stationary phase, low mass transfer impedance, high separation efficiency; high-pressure infusion pump to transport the mobile phase, short analysis time; widely used The high-sensitivity detector greatly improves the detection sensitivity. At present, different types of stationary phases, numerous separation modes and detectors, and the degree of automation have been widely used, so that the application of this method in drug testing continues to increase.

(Blum, et al. High performance liquid chromatography. British Journal of Hospital Medicine, 75(Sup2), C18–C21.(2014))

Instrumentation and Key Parts of HPLC Technology

High-Performance Liquid Chromatography relies on a well-designed set of instruments and components to deliver accurate and reproducible results. A standard HPLC system is composed of several key parts:

High-pressure pump：Provides a constant and stable flow of the mobile phase through the system, ensuring consistent separation efficiency. Modern pumps can deliver solvents at high pressures (up to hundreds of bars), which enables faster analyses with fine particle columns.

Injector or autosampler：Introduces the liquid sample into the mobile phase stream with high precision. Automated samplers improve throughput and reproducibility, which is crucial for drug analysis and pharmaceutical testing.

Chromatographic column：Packed with stationary phase particles, this is the core of the HPLC system. The column is responsible for separating compounds based on their polarity, size, or chemical interactions with the stationary phase.

Detectors：After separation, detectors such as UV-Vis, fluorescence, refractive index, or mass spectrometry (LC-MS) identify and quantify analytes with high sensitivity. The choice of detector depends on the type of compound and the analytical objective.

Data system and software：Integrated software collects and processes chromatographic signals, generates chromatograms, and provides quantitative analysis for reliable decision-making.

By understanding the instrumentation and HPLC machine parts, researchers can select the most suitable configuration for applications in medicine, pharmaceuticals, biotechnology, and drug development, ensuring both efficiency and accuracy in analytical workflows.

Applications of HPLC in Pharmaceutical Development

• Natural medicine analysis: The chemical components of natural medicines are complex, and there may be one or more active ingredients. This is difficult to control the quality of medicines and establish quality standards. HPLC can separate and identify the active ingredients of natural medicines, and then determine the content of active ingredients. The identification mode is established through fingerprint atlas to determine the quality of medicinal materials.
• Antibiotic analysis: Such as pazufloxacin mesylate, azithromycin, levofloxacin lactate, fleroxacin and other varieties of high-volume injection identification, content determination, determination of related substances, etc., all adopt HPLC methods. The HPLC method can avoid the strong antibacterial properties of antibiotics at high concentrations, as well as the long time and poor specificity of the microbial method.
• Related substance inspection: It is necessary to control the content of related substances in certain drugs during drug inspection. The quality of a drug is not only related to the content of the main ingredient of the drug, but also related to other ingredients in the drug except the main ingredient. The presence and amount of impurities are critical to the safety of the drug. In the formulation of drug quality standards, related substance inspection items are an important part. Because the HPLC method is simple, fast, exclusive, and accurate, it has become the mainstream method for detecting related substances. For example, the HPLC method for the determination of related substances in erythromycin lactobionate not only solves the problem that although the impurity spots can be detected in the TLC test, but cannot be accurately quantified, but also can accurately quantify erythromycin A, B, C components and related substances.
• Analysis of Chiral Drugs: Due to the same physical and chemical properties of drug enantiomers, it is difficult to separate them by conventional HPLC methods. For example, most amino acids have dextrorotatory and levorotatory forms, but often only a mixture of dextrorotatory and levorotatory forms (racemates) can be obtained. Chiral drugs often have the phenomenon that one enantiomer has strong biological activity and efficacy, while the other is ineffective or even toxic. Chiral high performance liquid chromatography can be used to resolve and quantitatively analyze chiral drugs. It has indirect method and direct method. The direct method does not require a derivatization reaction, and directly uses a chiral chromatography column or a chiral mobile phase for separation. The indirect method introduces an asymmetric (i.e., chiral) environment for the chiral stationary phase, so that the enantiomer (sample), the chiral substrate (such as the stationary phase) and the chiral source to be resolved form a diastereomeric molecule The complex.
• Identify drugs: In the HPLC method, the retention time is related to the structure and properties of the components, and is one of the qualitative parameters, which can be used to identify drugs. High-performance liquid chromatography-mass spectrometry (LC-MS) technology successfully combines the high separation capability of chromatography with the resolution capability of mass spectrometry, and has been widely used in the field of pharmaceutical qualitative research. In the United States Pharmacopoeia, the identification of Vasopressin uses this technology, by comparing the consistency of the ion peak mass-to-charge ratio of the test product and the reference product, so as to achieve the purpose of identification, and the method is highly specific.
• Drug impurity inspection: HPLC has high separation efficiency and sensitivity, and is widely used in drug impurity inspection. It is mainly used for the inspection of related substances in drugs. "Related substances" refer to synthetic raw material intermediates, by-products, degradation products and other substances that exist in drugs. The structure and properties of these substances are similar to the drugs, and their content is very low. Only by using chromatographic methods can they be separated and detected. For example, fluconazole, tinidazole and floxacin products are all identified by this method.
• Separation and purification of drugs: In the industrial production of genetic engineering products, the use of HPLC can effectively extract the products from the fermentation broth, thereby obtaining protein drugs and vaccine products with sufficiently high purity that are harmless to the human body. At present, apart from polymers, about 80% of drugs can be separated and purified with HPLC.
• In vivo drug analysis: Due to the rapid and accurate determination, wide range of mobile phase selection, high sensitivity (above 10-12g/mL), many types of packed columns, and many detectors to choose from, HPLC is a good in vivo laboratory research analytical method. Most drugs have UV absorption, so the most commonly used detector is the UV detector. Most drugs and endogenous substances are highly polar, and RP-HPLC chromatographic columns can be used to obtain a good separation effect by taking advantage of the difference in their polarities.
• Rapid clinical testing: Clinical analysis requires "short and fast", and sample inspection requires that it be completed in the shortest time. For this, HPLC has irreplaceable advantages. For example, in the analysis of amino acid samples, using a gradient HPLC-ODS column to analyze amino acids, an analysis can be completed within 1 hour.
• Provide basis for clinical dispensing: The application of HPLC provides a powerful weapon for the design of clinical medications. In recent years, HPLC has been used to determine the blood concentration of more than a dozen drugs. These data provide a reliable guarantee for guiding the clinical rational use of drugs and conducting pharmacokinetics research.

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Application of HPLC Technology in Drug Detection

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