Chromatography

Specimen Requirements and Procedure

Specimen requirements and procedures for clinical samples may vary depending on the type of chromatography and the analyte being assayed. Serum, plasma, whole blood, saliva, and urine samples are commonly used specimens.[15] All the clinical samples should be collected using an aseptic technique in appropriate containers. Clean-catch urine is obtained for urine samples. The urine sample should be free from any contamination, including blood. Similarly, serum or whole blood is collected in appropriate gel or anticoagulant tubes.[16] Samples should be handled carefully to avoid contamination or degradation. Processing should be carried out based on the chromatography technique used. This may involve extraction, derivitization, or dilution.[17] 

Dried blood spots (DBS) are collected for newborn screening from infants between 24 to 48 hours of age.[18] A heel prick specimen is preferred rather than from fingers or ear lobes.

Blood, tissue, or saliva is obtained to isolate DNA for nucleic acid assay. After the standard extraction technique, concentration and purity are measured by UV spectrometry or fluorometry.[19] Sample preparation steps like reverse transcription (for RNA) or polymerase chain reaction (PCR) amplification are performed before sample assay.

Diagnostic Tests

M. Tswett was the first to perform chromatography on plant pigments in 1906 using calcium carbonate as a stationary phase. Over time, thin-layer chromatography, gas chromatography, and HPLC were applied. Now, ultra HPLC, two-dimensional GC with full automation, and an advanced detector system are in use.[4] Since then, chromatography has evolved and found its application in many fields, such as analytical chemistry, clinical diagnostics, forensics, food science, pharmacology, and molecular biology.

Testing Procedures

The instrument used to perform a separation in chromatography is known as a chromatograph. For instance, in GC, the instrument is a gas chromatograph, and in LC, the instrument used is a liquid chromatograph. These instruments can respond to the amount of a compound that is exiting or eluting from the column as a function of the elution time or the volume of the mobile phase that has passed through the system.[1]

The following are the basic steps of chromatography.

1. Sample preparation: Sample preparation involves the extraction, purification, derivitization, and concentration of the analyte from the sample matrix. The preparation steps reduce any interferences during the assay.[16] Solid-phase extraction, liquid-liquid extraction, and protein precipitation are common. Filtration, centrifugation, and derivatization are followed after extraction for improved chromatographic separation.

2. Sample injection into the device:  In modern HPLC systems, an autosampler is commonly used for automated and precise sample injection.[24] The autosampler can handle multiple samples, perform injections with minimal user intervention, and ensure consistent results.

3. Separation of analyte: Separation is based on the different chromatography types. In gas chromatography, the analytes are vaporized and carried by the heated column, where they are separated based on their volatility and interaction with the stationary phase. The analytes are separated in liquid chromatography based on their size, charge, and polarity. In HPLC, the separation is similar to LC but at a higher pressure, leading to faster and higher separation resolution.[1] 

4. Elution of the separated substance: Elution is one of the important steps in chromatography that involves the collection of the compounds for analysis or further separation. The compounds elute from the columns at different times and concentrations. Extraction using solvents may be required.

5. Detection and interpretation: In column chromatography, as the solute migrates through the entire system and emerges from the column, the detectors continuously monitor the emerging mobile phase called the eluate and transmit signals in the form of voltage recorded on the chromatograph. Some of the detectors used in chromatography are mass spectrometry, refractive index, electrochemical, ultraviolet, flame ionization, electron capture, and fluorescence detectors.[25] Among these, ultraviolet detectors are the most commonly used in clinical laboratories.

Interfering Factors

Interfering factors during different stages of chromatography can affect the assay. Contaminants in the patient sample or during the sample preparation step might introduce unwanted contents and affect the detection. The presence of moisture or air bubbles in the samples might alter the retention time. Sample degradation after preparation and exposure to light, improper storage, handling, or extraction can alter the composition of analytes, affecting separation.[26] Overloading a GC machine with too much sample volume can cause the mixture to separate poorly. The column must be allowed to fully elute its contents before injecting the following sample.

Columns can be contaminated by precipitated proteins or by solvents. This will interfere with chromatogram results by causing distinct peaks or smearing to appear from previous elutions. Proper cleaning with solvents that dissolve the suspected contaminant is necessary to avoid this.[27] When making a column, the choice of adsorbent is key. The matrix of the column must be a loose, porous network permitting the entry and exit of macromolecules. Usually, silica is used, but this can vary.[28]

Changes in the pH of the buffer can affect the overall analysis. The pH changes after the sample injection when the sample and the mobile phase mix, mainly if the sample’s pH significantly differs from the mobile phase’s. Additionally, when air is exposed, a mobile phase can become acidic due to carbon dioxide absorption. Alterations in pH can affect the retention time (RT). Even a minor change of 0.1 pH units can change RT by as much as 10% with ionizable or ionic samples.[29] Buffer quality is also important for accurate results. Bacterial contamination in the buffer, precipitants, color change, and turbidity in the buffers can affect the run.

When similar drug molecules or metabolites are tested, the antibodies may bind to these related substances in the patient sample in a  non-specific manner, causing erroneous results. The structural similarities among steroid hormones, including differences in keto groups, double bonds, and OH groups, can lead to interference in affinity chromatography analysis, particularly when hormones with high serum concentrations (eg, cortisol) interfere with hormones of lower concentration but similar structures (eg,11-deoxycortisol). Similarly, high concentrations of dihydroxy epiandrosterone sulfate can interfere with estradiol, 17-OH progesterone, and testosterone. Furthermore, steroid metabolites contribute to the spectrum of potential interfering molecules.[30]

Results, Reporting, and Critical Findings

The chromatography results are interpreted from a chromatogram, a printout given by the detector. These results display the intensity of the detected signal (eg, absorbance, fluorescence, or mass spectrometry response) as a function of time or volume as the composites elute from the chromatographic column or stationary phase. Refer to Image 4.

The x-axis represents time (or volume) as the compounds elute from the column. The y-axis represents the signal intensity. The height of the peaks corresponds to the concentration of the separated compounds. The peaks represent the elution of the individual compounds. Each peak's shape, width, and area provide information about the compound's retention time, concentration, and purity.[31] Peak areas tend to provide a more precise means for measuring an analyte, whereas peak heights are easier to use if there is no complete resolution between the analyte and its neighboring peaks.[32] 

Retention time (RT) is the interval between the sample injection and the maximum peak. Each molecule has a characteristic RT under standard chromatographic techniques. The time taken for the specific component to elute is a characteristic feature. The baseline indicates the background signal and helps determine the noise level.[33] Peak width represents the efficiency of separation; the lesser the width, the more efficient the separation. The area under a peak represents the quantity of a compound eluted. Good resolution is indicated by better separation between 2 adjacent peaks. Higher resolution indicates better separation and distinct peaks. Retention volume is calculated from RT multiplied by the mobile phase's flow rate.[34] The compound can be identified by running reference material or control under the same operating conditions. Matching the retention time and the component peak with a standard or control identifies the unknown component in the sample.

Clinical Significance

Chromatography helps accurately separate, identify, and quantify compounds in patient samples, enabling diagnostic, therapeutic, and research applications in healthcare.

Newborn Screening of Metabolic Disorders

Newborn screening using heel prick dried blood spots is a valuable tool for detecting various inborn errors of metabolism using chromatography. Thin layer chromatography, HPLC, LC-MS, and GC-MS-based techniques are commonly employed to assay various analytes. Commonly screened disorders include phenylketonuria, maple syrup urine disease, homocystinuria, tyrosinemia, methyl malonyl acidemia, homocystinuria, citrullinemia, tyrosinemia type I, carnitine deficiency, fatty acid oxidation disorders, and lysosomal storage disorders, etc. Most countries have implemented these screening programs, but the adoption and extent may vary from country to country.[35] This allows for the early detection of certain genetic and metabolic disorders, enabling timely intervention and treatment before disability. 

Diagnosis of Hemoglobinopathies, Thalassemia, and Porphyrias

HPLC offers high sensitivity and specificity compared to electrophoresis for diagnosing hemoglobinopathies and thalassemias. Cation exchange HPLC is the method of choice that can separate nearly 45 different hemoglobin variants and accurately quantify normal hemoglobin.[22] It is also used for the newborn screening of sickle cell anemia. Denaturing HPLC is also used to detect alpha and beta globin chain mutations in thalassemia in chorionic villi samples. Analysis of porphyrins like 5-aminolevulinic acid (ALA) and porphobilinogen (PBG) in physiological liquids can be performed by LC/MS to diagnose the type of porphyrias.

Diabetes Mellitus and Other Metabolic Disorders

The hemoglobin A1c (HbA1c) is one of the gold standard tests for diagnosing diabetes mellitus and is measured using high-performance liquid chromatography (HPLC). Among the National Glycohemoglobin Standardization Program (NGSP) recommended methods for HbA1c, HPLC offers the least interference, high accuracy, and traceability. In the pediatric age group, it is used to quantify different steroids and aids in the diagnosis of congenital adrenal hyperplasia, hyperaldosteronism, Cushing's disease, and Addison's disease. LC-MS is more accurate than conventional immunoassay for diagnosing the above conditions.[36]

Cancers

Denaturing HPLC detects BRCA1 and BRCA2 gene mutations, guiding risk assessment and management for hereditary breast cancer.[37] Clinically important biogenic amines are bioactive endogenous compounds that play a significant physiological role. Serotonin, dopamine, noradrenaline, adrenaline and their metabolites, vanillylmandelic acid, and 5-hydroxy indoleacetic acid (5-HIAA) acid are some of the biogenic amines that are biomarkers for the diagnosis, prognosis, therapy, and follow-up of several neuroendocrine and cardiovascular diseases.[38] 

Therapeutic Drug Monitoring

This involves quantitatively measuring the drug in biological fluids to manage drug therapy. It also involves assessing the efficacy and safety of a drug in the treatment. Mass spectrometric methods for many classes, such as immunosuppressive drugs, antivirals, antiepileptic drugs, chemotherapeutic drugs, antibiotics, antidepressants, and antipsychotics, are employed along with chromatography for drug monitoring.[14]

Genetic Analysis

Denaturing HPLC is a sensitive and comprehensive technology for studying gene expressions and detecting many gene mutations and polymorphisms. Single-stranded DNA is subjected to varying temperatures in a reversed-phase HPLC column, which will melt or denature at different temperatures based on the nucleotide sequence.[19] The main advantage is that up to 1.5 kb fragment can be analyzed in 5 minutes. Some applications are detecting mutations in the CFTR gene for cystic fibrosis, globin gene mutation in thalassemia, mutations related to phenylketonuria, and other metabolic disorders.

Toxicology

Liquid chromatography-mass spectrometry is used in the characterization of urine samples in patients with suspected benzodiazepine use disorder. Chromatography can be utilized to confirm the diagnosis of anabolic steroid use disorder through pulverization of nail clippings and urine samples at a level of pg/mL.[39] Thus, chromatography is an extremely sensitive technique that can potentially be used to investigate athletes' doping.[40]

Quality Control and Lab Safety

Quality control and laboratory safety are paramount when running chromatography for medical diagnostics.[41] Good laboratory practice and quality assurance systems ISO 9001 and ISO 17025 have established many rules to install and maintain a quality system in laboratory diagnostics.[42] The initial step in quality control involves using a validated method to meet analytical requirements. At the same time, routine analysis necessitates assessing performance based on relevant tests such as calibration injections and peak shape observations to ensure instrument functionality and proper separation.

Analytical accuracy is typically assessed through reference materials, internal reference samples, and participation in proficiency testing to ensure quality performance. Whether the sample is urine or blood, an internal standard or a substance similar to the substance of interest (analyte) must be set in the GC-MS. For example, if morphine is the analyte, nalorphine may be used as an internal standard.[43] Regular calibration of instruments, proper sample handling, and meticulous record-keeping are essential to maintain precision.

Chromatography involves the usage of potentially caustic skin irritants such as ethanol or NaOH. Care must be taken in advance to ensure minimal exposure to biological substances and irritation.[44] 

A material safety data sheet (MSDS) is a catalog of chemicals and their potential risks of use.[45] At a minimum, healthcare facilities must have an MSDS that notes the physical risks, symptoms of exposure, safe handling instructions, suggested personal protective equipment, the names of all hazardous ingredients, characteristics of the reagents, routes of entry, exposure time limits, emergency and first aid instructions, the contact information of the manufacturers, as well as dates of MSDS preparation, per Occupational Safety and Health Administration (OSHA) guidelines.[46] When onboarding new healthcare professionals to a laboratory environment, they should be allocated adequate time to orient themselves to the chemicals they will utilize. 

Whether it is a blood or urine sample, samples are exchanged between the hands of assistants and laboratory staff, which merits the utmost caution in preventing exposure and contamination.[44] Healthcare staff is encouraged to hold each other accountable for maintaining aseptic techniques to achieve clean samples.[47] Standard safety precautions should be followed when placing and securing the gas cylinder or mobile phase source used for gas chromatography (GC). If hydrogen is used as the carrier gas, extra precautions should be taken in training laboratory personnel in the handling and use of this combustible and potentially explosive gas. Proper ventilation facilities should be available in the work area to deal with carrier gas that has passed through the GC system.[48] All samples, reagents, and solutions used during a GC analysis should also be handled and stored using appropriate laboratory procedures.[49]

Enhancing Healthcare Team Outcomes

Healthcare team outcomes can be enhanced when using chromatography for medical diagnostics by ensuring effective interdisciplinary communication and proper training for all team members and implementing strict quality control measures. In the preanalytical phase, clinicians outside the laboratory, especially the medical assistants, residents, and nurses, must work with laboratory scientists to obtain appropriate samples, correctly handle the samples, record patient information, and maintain specimen integrity. Effective communication between healthcare professionals and the laboratory team ensures a clear understanding of sample requirements and any considerations. Reporting adverse events or issues during sample collection helps the laboratory assess the impact on results.

Technicians should be proficient in handling chromatography techniques and instrument operation in the laboratory. They should be backed by professionals with a background in inherited metabolic diseases and drug kinetics to evaluate the results, confirm the diagnosis, and advise on further action plans. All the lab professionals should be involved in the comprehensive plan of selecting the appropriate chromatographic methods, deciding on the sampling requirements, quality control, and reporting. International guidelines for assessing laboratory performance, such as ERNDIM and CDC, are available. Patient confidentiality, informed consent, and accurate reporting of results should be ensured throughout the process. By integrating these aspects, healthcare professionals can ensure that chromatography in medical diagnostics contributes effectively to patient-centered care, improved outcomes, and enhanced safety.