CN102692396B - Endotoxin detection system and quantitative detection method thereof - Google Patents

Abstract

The invention discloses an endotoxin detection system and a quantitative detection method thereof, belonging to the field of endotoxin monitoring. The system integrates the optical path detection system on the basis of a conventional laser particle size detector. It uses dual laser sources for irradiation. The output light enters the detection pool through the optical path and is received by a multi-point scattered light receiver, and then through photoelectric conversion and signal amplification. After processing, it enters into the quantitative calculator, which, according to the difference in the scattering intensity of endotoxin particles at more than three different angles, fits the correlation between the astigmatism and the endotoxin concentration, calculates the endotoxin concentration and displays it, when the endotoxin concentration exceeds The limit value triggers the early warning system and realizes online monitoring. The invention can realize quantitative detection of endotoxin, has high detection sensitivity, does not consume reagents, has no pollution to liquid medicine, has short detection time and high reliability.

Description

A kind of endotoxin detection system and its quantitative detection method

technical field

The invention relates to an endotoxin detection technology, in particular to an endotoxin detection system based on laser particle size detection and a quantitative detection method thereof, belonging to the field of endotoxin monitoring.

Background technique

Bacterial endotoxin is lipopolysaccharide, also known as liposome, which is a component of the outer cell wall of Gram-negative bacteria and widely exists in nature. When this substance enters the human blood, it will cause fever, commonly known as pyrogen reaction. Because such substances may cause serious adverse reactions in humans, strict control is required in drug injections.

At present, endotoxins cannot be monitored online during the production process of water for injection and injections, and the detection of endotoxins still relies on the limulus reagent method or rabbit method for inspection after production. These methods and processes not only have low detection sensitivity, but once the endotoxin in the product is found to exceed the standard, the entire batch of products will be disposed of as waste, which will not only bring economic losses to the enterprise, but also bring huge clinical applications to the enterprise risk. Therefore, in order to ensure the quality and safety of water for injection and injections, prevent sudden pollution during the production process, monitor the integrity of the ultrafiltration membrane during production, and realize online monitoring of endotoxins is of great significance.

At present, the laser particle size detector has not been applied to the detection of endotoxin. We analyze the highly sensitive Malvern Nano ZSZEW3600 laser particle size analyzer, which can detect the particles in the solution with the endotoxin concentration above 10EU/ml. This cannot meet the detection concentration of endotoxin in injection. The laser particle size detector is based on the different astigmatism signals of the particles at different angles, and performs particle size analysis on the multi-point astigmatism signals of more than two points, but it cannot detect the particle concentration, so it cannot be used for the quantitative detection of endotoxin.

Contents of the invention

Aiming at the defects in the prior art, the present invention proposes an endotoxin detection system with high detection sensitivity and capable of realizing on-line detection and a quantitative detection method thereof.

The endotoxin detection system includes a laser source, a short-focus lens, a grating, a long-focus lens, a detection cell, a scattered light receiver, a photoelectric converter, a signal amplifier, a signal processor, a data collector, a quantitative calculator, a data display and an early warning The device, wherein: the output light of the laser source passes through the short-focus lens, the grating and the long-focus lens in turn and then enters the detection pool, the light transmitted from the detection pool is received by the scattered light receiver and enters the photoelectric converter, and the output signal of the photoelectric converter After passing through the signal amplifier, signal processor and data collector, it enters the quantitative calculator to calculate the endotoxin concentration, and the output of the quantitative calculator is connected to the data display and the early warning device.

The quantitative detection method of the endotoxin detection system is: detect the scattered light signal intensity of endotoxin particles in the solution at more than three different angles, according to the light scattering formula I=K·C·f(D)·f(E), by Among them, f(D) and f(E) are calculated from the scattered light signals of any two angles, and the light scattering formula is simplified to S=f C+g, and then the endotoxin concentration of the solution is calculated by the scattered light signals of other angles , to achieve quantitative detection;

In the above formula: I is the scattered light signal intensity; K is the proportional constant; C is the endotoxin concentration; f(D) is the particle size parameter function; f(E) is the scattering spectrum function; The ln linearization data processing of I is obtained; the correlation coefficient f=K·f(D)·f(E); g is the correction parameter.

Technical effect:

1. The detection does not consume reagents, does not cause any pollution to the liquid medicine, and is economical and environmentally friendly.

2. The optical path system is integrated, which improves the detection sensitivity and can detect endotoxin solutions above 0.5EU/ml.

3. Combined with the quantitative calculation method of multi-point astigmatism signals of more than three points, the quantitative detection of endotoxin is realized.

4. The detection mode is divided into two types: online detection and offline detection, with flexible application, good practicability, and short detection time, which can meet the online monitoring requirements of water for injection, injection and ultrafiltration membrane integrity.

5. An early warning system is added, which can trigger an early warning when the endotoxin concentration of the solution exceeds the standard, and realize online monitoring with high reliability.

Description of drawings

Fig. 1 is the composition structure schematic diagram of the system of the present invention, label name among the figure: 1, laser source; 2, short-focus lens; 3, grating; 4, long-focus lens; 5, detection pool; , photoelectric converter; 8, signal amplifier; 9, signal processor; 10, data collector; 11, quantitative calculator; 12, data display; 13, early warning device.

Figure 2 is a schematic diagram of the principle of quantitative calculation of endotoxin.

Fig. 3 is a detection and early warning flow chart of the system of the present invention.

Detailed ways

The present invention will be further described below.

The structure of the endotoxin detection system of the present invention is shown in Figure 1, comprising a laser source 1, a short-focus lens 2, a grating 3, a long-focus lens 4, a detection cell 5, a scattered light receiver 6, a photoelectric converter 7, and a signal amplifier 8 , signal processor 9, data collector 10, quantitative arithmetic unit 11, data display 12 and early warning device 13, wherein: the output light of laser source 1 enters detection pool after passing through short focus lens 2, grating 3 and long focus lens 4 successively 5. The transmitted light from the detection cell 5 is received by the scattered light receiver 6 and enters the photoelectric converter 7, and the output signal of the photoelectric converter 7 passes through the signal amplifier 8, the signal processor 9 and the data collector 10 in turn and then enters the quantitative calculator 11 calculates the endotoxin concentration, and the output of the quantitative calculator 11 is connected to the data display 12 and the early warning device 13 .

The present invention integrates the optical path system on the basis of the conventional laser particle size detector, and preferably detects the scattering optical path at about 90°. Laser source 1 is a double-beam laser source, using FLS-2600B laser source from Canada EXFO Company; short-focus lens 2 adopts ultra-low dispersion lens; grating 3 adopts fiber coupler type fiber grating; long-focus lens 4 adopts low-consumption NV-202m lens. The detection pool 5 is made of optical glass, including two types of online detection pool and offline detection pool: the upper and lower ends of the online detection pool are provided with liquid outlets and liquid inlets, and the online detection pool is connected to the A loop is formed in the pipeline of the injection production line to realize the continuous detection of the liquid medicine. Electromagnetic valves are installed on the liquid outlet and the liquid inlet, and an ultrasonic probe is also installed at the liquid inlet to eliminate air bubble interference; offline detection The pool is a semi-sealed detachable detection pool, and the top of the detection pool is equipped with a liquid filling port for manual replacement of solutions. The scattered light receiver 6 adopts the Picometrix LLC high-speed multi-point wide-angle scattered light receiver module of API Company, which is arranged in a multi-point interval logarithmically; the photoelectric converter 7 adopts the HESMC photoelectric converter of Hengqi Electronics Co., Ltd. of the United States; the signal amplifier 8 It is composed of an amplifier circuit and a filter circuit, among which the amplifier circuit consists of three ICL7650 amplifiers connected into a differential amplifier circuit, and the filter is a commonly used RC network; the signal processor 9 adopts the CONVERTER-VLC.602 programmable signal of Italy VAL.CO Processor; data collector 10 adopts A/D converter Max132 of Maxim Company. Quantitative calculator 11 is independently developed by the unit. The calculation principle of the quantitative calculator is shown in Figure 2. According to the light scattering formula I=K·b·C·f(D)·f(E)·f(Δλ), the formula Middle: I is the scattered light signal intensity, K is the proportional constant, b is the thickness of the solution to be tested, C is the endotoxin concentration (unit: EU/ml), f(D) is the particle size parameter function, f(E) is Scattering spectrum function, f(Δλ) is a wavelength function, the thickness of the solution to be measured (colorimetric cell) is fixed, and the wavelength is fixed, so b and f(Δλ) are constants, and the light scattering formula can be simplified as I=K·C·f( D) f(E). The constant K value can be obtained by detecting endotoxin standard solutions with different concentrations. The system detects the scattered light signal intensity of endotoxin particles in the solution at more than three different angles. According to I=K·C·f(D)·f(E ), calculate f(D) and f(E) from the scattered light signals at any two angles, and simplify the light scattering formula to S=f C+g, where: S is the degree of astigmatism, and S is derived from I The ln linearization data processing is obtained, the correlation coefficient f=K·f(D)·f(E), g is the correction parameter, and then the endotoxin content in the solution is calculated by the scattered light signals of other angles, and the quantitative detection is realized. The data display 12 adopts the 128×64 dot matrix display terminal of Jinchuangdao Company, which is used to display the detected endotoxin concentration value; the early warning device 13 adopts the integrated achievement electronic CMS7000-500 alarm monitoring software and piezoelectric early warning system. The detection and early warning process is shown in Figure 3. The endotoxin concentration limit can be set in advance in the early warning software. When the endotoxin concentration in the detected liquid exceeds the limit, the alarm will alarm.

The process of using the online detection in the present invention is as follows: according to the production needs, preset the endotoxin concentration limit, the coefficient f and the parameter g in the quantitative calculator, start the production line, and let the solution enter the online detection pool, according to S=f·C +g calculates the endotoxin concentration, when the concentration is lower than the limit value, the system keeps running, and when the concentration is higher than the limit value, the early warning system alarms.

The operation steps of the off-line detection of the present invention are as follows: preset the coefficient f and the parameter g in the quantitative calculator, take out the off-line detection pool, clean it with pure water, rinse it with the solution to be tested for 2 to 3 times, and then add the solution, put the detection cell in the system, and calculate the endotoxin concentration according to S=f·C+g.

The following provides an example of off-line quantitative detection of endotoxin in Danshen infusion:

Replace the off-line detection pool, take the working standard endotoxin, prepare a standard solution with a concentration of 100EU/ml with the test water, and then use the test water to gradually dilute to a series of standard solutions with a concentration of 0.5, 1, 5, 10. 50EU/ml, put the solution in the detection cell for detection, and perform data regression calculation through the quantitative calculator. The linear equation is: S=0.056C+0.165, R ² =0.997. The calculation results are shown in Table 1.

Table 1

The intermediate of salvia miltiorrhiza infusion (provided by Shanghai Huayuan Anhui Jinhui Pharmaceutical Co., Ltd., batch number: 05110209) was used to calculate the endotoxin content by this system detection method and the turbidimetric method of Limulus reagent, and the results are shown in Table 2.

Table 2

The results show that the endotoxin content measured by this system is close to that of the turbidimetric method of Limulus reagent. At present, the limulus reagent turbidity method has been included in the Chinese Pharmacopoeia, and the measurement results are relatively accurate. The results measured by this system are similar to it, which also shows the reliability of this system.

The following provides an example of online quantitative detection of endotoxin in Danshen infusion:

Take two parts of Danshen infusion, one is the normal drug solution (provided by Shanghai Huayuan Anhui Jinhui Pharmaceutical Co., Ltd., batch number: 10011605), and the other is the contaminated drug solution, and carry out online monitoring on the two drugs respectively . Set the detection parameters: f is 0.056, g is 0.165, the endotoxin limit is 1EU/mL, the production line is turned on for detection, and the endotoxin concentration is calculated by the regression equation S=0.056C+0.165. The results are shown in Table 3.

table 3

The results show that when the endotoxin pollution exceeds the limit value, the system can give an alarm, which can meet the quantitative monitoring of the endotoxin pollution in the production of injections.

Claims (2)

1. An endotoxin detection system for drug injections, characterized in that it includes a laser source (1), a short-focus lens (2), a grating (3), a long-focus lens (4), a detection cell (5), Scattered light receiver (6), photoelectric converter (7), signal amplifier (8), signal processor (9), data collector (10), quantitative calculator (11), data display (12) and early warning device (13), wherein: the output light of the laser source (1) enters the detection pool (5) after passing through the short-focus lens (2), the grating (3) and the long-focus lens (4) sequentially, and the output light of the detection pool (5) The light is received by the scattered light receiver (6) and enters the photoelectric converter (7), and the output signal of the photoelectric converter (7) passes through the signal amplifier (8), signal processor (9) and data collector (10) in sequence Enter the quantitative calculator (11) to calculate the endotoxin concentration, the output of the quantitative calculator (11) is connected to the data display (12) and the early warning device (13); the laser source (1) adopts a double-beam laser source; the detection The pool (5) is made of optical glass, and the detection pool (5) includes an online detection pool and an offline detection pool; the online detection pool is provided with a liquid outlet and a liquid inlet, and the online detection pool passes through the liquid outlet and the liquid inlet The outlet is connected to the pipeline of the injection production line, the liquid outlet and the liquid inlet are equipped with electromagnetic valves, and the liquid inlet is also equipped with an ultrasonic probe for eliminating air bubble interference; the offline detection pool is semi-sealed and detachable Type detection pool, the liquid filling port is set on the top of the detection pool; the scattered light receiver (6) adopts more than three points of multi-point wide-angle scattered light receiver; the grating (3) is a fiber-coupled fiber grating; The signal amplifier (8) is composed of an amplification circuit and a filter circuit; the endotoxin detection system uses a 90° scattered light path for detection. 2. A quantitative detection method based on the endotoxin detection system for drug injections according to claim 1, characterized in that: The method comprises the following steps: detecting the intensity of scattered light signals of endotoxin particles in solution at more than three different angles, according to the light scattering formula I=K·C·f(D)·f(E), any two angles Calculate f(D) and f(E) from the scattered light signal, simplify the light scattering formula to S=f C+g, and calculate the endotoxin concentration of the solution by using the scattered light signal at other angles to achieve quantitative detection; In the above formula: I is the scattered light signal intensity; K is the proportional constant; C is the endotoxin concentration; f(D) is the particle size parameter function; f(E) is the scattering spectrum function; The ln linearization data processing of I is obtained; the correlation coefficient f=K·f(D)·f(E); g is the correction parameter.

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