Instrumax CAD-tobramycin Assay Determination-Application Note

1, Preface

Charged Aerosol Detector (CAD)(charged aerosol detector,CAD) is a new general-purpose detector developed in recent years. Its detection has nothing to do with the molecular structure of the substance being measured, and the substance being measured does not need to be ionized. It can be used as long as the substance being measured is a semi-volatile or non-volatile compound.CAD detection. At the same time,CAD It is a mass-sensitive detector. The response value of the substance being measured is determined by the quality of the sample being tested. As long as the quality of the sample being tested is consistent, the measured response value will basically be the same, and a lower detection limit and higher sensitivity can be obtained.

Charged Aerosol Detector (CAD) Yes INSTRUMAX The R&D team has achieved major scientific and technological achievements after years of painstaking research and repeated experiments. The detector uses advanced electricity and atomization technology to achieve high-sensitivity and high-precision detection of trace samples.

tobramycin(Tobramycin), also known as tobramycin, is an aminoglycoside antibiotic. Aminoglycoside antibiotics do not contain conjugated structures and have weak UV absorption, making them unsuitable for conventional analysis using UV detectors directly.2020 Edition of "Chinese Pharmacopoeia"tobramycin Materials related to raw materials are detected using evaporative light scattering detectors (ELSD),ELSD with CAD Compared with other methods, the sensitivity is low and the repeatability is poor; the content detection item uses a microbial assay method, and the specificity of the method is poor. 《USP》In current standards tobramycin The detection of relevant substances uses thin layer chromatography, which cannot conduct in-depth analysis of each impurity; the content detection method is to detect the sample through a UV detector after derivatization, and the operation is relatively cumbersome. For this reason, the United States Pharmacopeia Commission 2023 year 1 Draft for comments released monthly^[1], adopt HPLC-CAD Method testing tobramycin related substances and contents.

This article refers to the consultation draft method and adopts INSTRUMAX CAD Combined with Agilent SB-C18 bar pair tobramycin The content determination method was investigated. This method has strong specificity, high sensitivity, good repeatability, good linearity and can meet the requirements of tobramycin Quality control requirements for raw materials and related preparations.

2, instruments and materials

Instruments:Agilent1200 liquid chromatograph+Charged Aerosol Detector (CAD)(INSTRUMAX, Model: CADetector a1)

Reagents: pure water (Watson's distilled water), acetonitrile (chromatography grade), trifluoroacetic acid (analytical grade)

Sample:tobramycin Samples (kindly provided by user teachers)

3, methods and results

2.1 Chromatographic conditions

Column: Agilent ZORBAX SB C18,4.6mm x250mm，3.5μm

Column temperature:10℃ Flow rate:0.8mL/min

Injection volume:25μL

Mobile phase A:0.3%TFA Aqueous solution mobile phase B:0.3%TFA Acetonitrile solution

gradient elution

CAD Conditions:

Instrument model:INSTRUMAX CADetector a1

Drift tube temperature:40℃ Atomized air flow:3L/min

Corona current:1μA Corona airflow:1L/min

Gain:0.2

2.2 Sample preparation

(1) Blank solvent: mobile phase A

(2)Control solution-That is, the sample solution prepared from raw materials

Reference substance stock solution: take tobramycin Reference substance approx. 18mg(0.018g), accurately weighed, set 100m1 In the measuring flask, add diluent (mobile phase A) for approx.10ml Dissolve and dilute to volume, shake well.The concentration is 180.0μg/mL

linear solution 1: Precisely measure the content reference substance stock solution 3ml, set 50m1 In a measuring bottle, dilute to the mark with diluent and shake well.The concentration is 10.8μg/mL

linear solution 2: Precisely measure the content reference substance stock solution 2ml, set 25m1 In a measuring bottle, dilute to the mark with diluent and shake well.The concentration is 14.4μg/mL

linear solution 3: Precisely measure the content reference substance stock solution 1m1, set 10m1 In a measuring bottle, dilute to the mark with diluent and shake well.The concentration is 18.0μg/mL

linear solution 4: Precisely measure the content reference substance stock solution 3ml, set 25m1 In a measuring bottle, dilute to the mark with diluent and shake well.The concentration is 21.6μg/mL

linear solution 5: Precisely measure the content reference substance stock solution 3ml, set 20m1 In a measuring bottle, dilute to the mark with diluent and shake well.The concentration is 27.0μg/mL

2.3 Experimental results

2.3.1 Exclusiveness

The blank does not interfere with the determination of the main peak and has good specificity.

Figure 1 Blank gradient (no injection)

Figure 2 Blank solvent (mobile phase A)

Figure 3 tobramycin Control stock solution 180μg/mL Figure

2.3.1 linear solution 1-Number of theoretical boards

linear solution 1(The concentration is 10.8μg/mL), calculate the number of theoretical plates (USP), the number of theoretical boards is 16600.5(＞3000), the column performance is good.

Figure 4 tobramycin Reference linear solution 1(10.8μg/mL) diagram

2.3.2 Reference substance injection precision

The concentration is 18.0μg/mL of reference solution, continuous injection 5 needle, peak area RSD% is 0.980%(<2.0%), with good precision.

2.3.3 Reference solution linearity

linear solution 1 to linear solution 5, inject samples separately 1 Needle, use linear solution concentration and peak area to make a linear regression equation.

The results show that the linear correlation coefficient R is 0.99918(＞0.995), the linearity is good.

1. Experimental discussion:
2. It can be seen from the experimental results that the blank gradient is 15~16min There are gradient peaks, which have no impact on the experiment; the blank solvent is in 2~5min Yes 4 peak, because it is related to tobramycin The retention times of the main peaks are far apart and do not interfere with the detection, so no further investigation and investigation were conducted in this experiment.
3. This experiment examined several aspects such as specificity, raw material injection precision, linearity, preparation samples, and preparation injection precision.Instrumax CAD For the suitability of the sample, the results show that Instrumax CAD The sample detection, repeatability, linearity and other results are good and can meet the experimental needs.

References[1] USP-NF Draft for comments Tobramycin [S].