Biological Analysis

Methods for the determination of Plasma Concentration

Plasma Concentration refers to the total concentration of drugs in plasma after absorption, including drugs bound to plasma proteins or free in plasma, and sometimes also refers to the concentration of drugs in whole blood. The intensity of drug action is proportional to the concentration of the drug in the plasma, and the concentration of the drug in the body varies with time. The monitoring method is mainly high performance liquid chromatography.

Plasma Concentration

1. Definition

Plasma Concentration refers to the total concentration of the drug in the plasma after absorption, including the drug bound to plasma proteins or free in the plasma, and sometimes it can also refer to the concentration of the drug in whole blood. The intensity of drug action is proportional to the concentration of the drug in the plasma, and the concentration of the drug in the body varies with time.

2. Clinical significance

In clinical practice, it is often found that the same dose of drugs is used for different patients with the same disease, and the efficacy is often very different. It gets better; some don’t. The toxic and side effects are also different, some have almost no side effects or mild; some are severely poisoned. This is because of individual differences (age, gender, physical condition, genetics, ethnicity, etc.). For example, in the treatment of convulsions and cardiac arrhythmias with phenytoin, the optimal therapeutic dose varies widely in different individuals. When treated with the usual dose, the blood concentration of the drug varies greatly. Some people are as low as 2 mg/L, which has no effect at all; some people are as high as 50 mg/L, and the patient is seriously poisoned.
In order to achieve rational drug use, scientists have done a lot of experiments and found such a meaningful phenomenon: different people, their effective drug doses vary greatly, but their safe and effective Plasma Concentration vary less. Usually less than 1 times. When the Plasma Concentration is greater than the safe and effective range, its side effects and manifestations and its degree vary little in different patients. Take the example of phenytoin again, the safe and effective blood concentration range of this drug is 10-20 mg/L in almost all patients. Similarly, when the blood concentration of phenytoin exceeds the safe range, almost all patients have toxic reactions. When the blood concentration is 20 to 30 mg/L, the patient appears mentally abnormal.
For those drugs whose effective concentration is close to the toxic blood concentration (such as digitalis), as well as for those patients taking long-term or concomitant medication, in order to prevent the toxic and side effects of the drug from being too high in the body, the blood concentration should be measured frequently.
Nowadays, some qualified hospitals not only master the dosage of drugs and the number of times of administration, but also often measure the Plasma Concentration to guide the clinical selection of the best treatment plan and the most suitable treatment dose for different individuals.

3. Monitoring methods

At present, there are many methods for the determination of Plasma Concentration, and the most commonly used methods are spectroscopy, chromatography, and immunoassay.

1) Spectroscopy

Such methods include colorimetry, UV-Vis spectrophotometry, and fluorescence methods. The sensitivity of this method is relatively low, and it can only monitor drugs with a concentration of more than 1.0 μg·L-1 in body fluids, and metabolites or compounds with similar structures interfere greatly with the monitoring. In the searched literature, the minimum detection limit of aminophylline concentration in blood by dual-wavelength ultraviolet spectrophotometry is 0.5 μg·mL-1, and the minimum detection limit of phenytoin sodium is 5 μg·mL-1. Although differential spectrophotometry, derivative spectrophotometry, etc. are used to improve the specificity of the method and reduce the interference of impurities, it cannot improve the sensitivity of this type of detection method. In addition, the application of such methods requires a large amount of sample, especially for differential spectrophotometry. The above shortcomings limit the application of such methods in Plasma Concentration monitoring. Especially with the development of chromatography technology in recent years, the application of spectrometry has gradually decreased due to its limitations in sensitivity and specificity. However, for body fluid samples with high drug concentration or samples with large sample volume, this type of method is still an economical, simple and fast monitoring method.

2) Chromatography

Chromatography is currently the fastest growing and most applicable method. The biggest advantage of this method over spectroscopic methods is that it not only has superior quantitative effect, but also has the effect of separating multiple samples at one time. In addition, this method is highly sensitive, and the concentration of drugs that can be measured can reach 0.001-1 μg·mL- 1. Especially in recent years, the development of mass spectrometry has expanded the monitoring range, reaching 1.0×10-9 to 1.0×-6 μg·mL-1 or even lower.

(1) High performance liquid chromatography (HPLC) method
HPLC method is currently the most commonly used method for monitoring blood concentration in clinical practice. In the literatures examined, the use of HPLC method to monitor blood concentration of antiepileptic drugs reaches more than 80% in all methods. This method has high detection sensitivity, high precision and strong specificity. Moreover, the HPLC method has a wide range of applications and can be used in many fields of drug research. It has become more and more popular, but this method has very high requirements for the pretreatment of samples, mainly because the chromatographic column is an important carrier for separation and analysis of samples. The purity of the samples to be analyzed is very high, and macromolecular proteins and other macromolecular substances must be completely processed to minimize the impact on the column efficiency of the analytical column and prolong the use time of the chromatographic column.
HPLC operation is time-consuming and relatively expensive. At present, in view of the tedious problem of sample processing in chromatographic and spectroscopic analysis, some chromatographers have successfully developed a packed column for online purification and enrichment of biological fluids. No pretreatment is required during sample monitoring, and macromolecular substances such as proteins are eluted. It is not retained, and small molecules can freely enter and exit the hydrophobic part of the stationary phase for analysis. This method saves time and effort. In recent years, SPE columns have been widely used in HPLC systems at home and abroad, which greatly simplifies the method of body fluid analysis, but this process increases the cost of sample monitoring. The development of liquid-mass spectrometry technology provides a broad space for sample analysis, and the combination of mass spectrometry provides more sensitive and accurate technical support for the further development of chromatography, and greatly expands the application of high performance liquid chromatography. For example, when monitoring the concentration of telmisartan in serum, the minimum detection limit is 20 μg·L-1 when monitoring by high performance liquid chromatography alone, but when monitoring by liquid-mass spectrometry, the sensitivity can be improved to 0.5μg·L-1.

(2) Gas chromatography (GC) method
The GC method has high selectivity, high sensitivity, less sampling, fast analysis speed, wide application range, and can prepare high-purity substances. The disadvantage of gas chromatography is that the measured components or their derivatives must have certain volatility and thermal stability, so samples with thermal instability or high polarity cannot be monitored by GC. At present, with the development of stationary phase and the application of derivatization technology, the monitoring of many samples is no longer limited. However, the application of gas chromatograph in in vivo drug analysis is relatively rare.

(3) Thin-layer chromatography
Thin-layer chromatography is a chromatographic method that separates samples on a chromatographic plate and then quantifies the separated samples. The sensitivity and reproducibility of this method are worse than HPLC and GC methods, and there are many operation steps, which is limited in practical application. However, this method is simple to operate and can meet the requirements for concentration monitoring of general drugs. In addition, the high-efficiency capillary electrophoresis method developed at the end of the 20th century has gradually attracted attention in drug analysis and in vivo drug analysis due to its high-efficiency separation technology and high sensitivity.

3) Immunization

Currently, the monitoring of drug concentration by immunoassay is mainly based on the principle of protein competition. From the current literature, radioimmunoassay, enzyme immunoassay, and fluorescence immunoassay are the most commonly used methods for monitoring Plasma Concentration. This method is mainly based on EMIT, which has simple sample processing and short time to obtain results, and has become an important basis for clinical evaluation of curative effect. Most of these instruments are developed abroad, are expensive, and are limited to the drugs that can be monitored after they are developed. However, this type of instrument still attracts clinical attention due to its rapid acquisition of results, simple sample processing and high sensitivity.

Blood to Plasma Ratio