Determination of residual vulcanizing agent in the

Determination of residual sulfides in antisense oligonucleotides

antisense oligonucleotides are antisense oligonucleotides, whose nucleotide sequences can complement target RNA or target DNA, inhibit or block gene transcription and expression, or induce RNaseH to recognize and cut mRNA, so that it loses its function. Antisense nucleic acid drugs are a new field or revolution in pharmacology. They are the most accurate, efficient and widely significant potential strategies for regulating specific gene expression. They can be used as research tools for the study of the physiological functions of some specific proteins and genes, and as therapeutic drugs for the treatment of many diseases such as cancer, infectious diseases and so on. However, the function of antisense oligonucleotides largely depends on their stability, bioavailability and binding characteristics with target genes. These physical and chemical properties can be changed by specific chemical modification, so as to increase the anti-virus, anti-tumor and other specific gene expression inhibitory activities. Phosphorothioate modified thiooligonucleotides (the non bridged oxygen atom in the phosphodiester skeleton is replaced by sulfur) are the most widely studied antisense nucleic acids. Now almost all ODNs entering the clinic are PS-ODN [2]. It has the characteristics of anti nuclease hydrolysis, good nucleophilicity, convenient isotope labeling and so on

common thio modifiers include beauage reagent (3h-1,2-benzodithiol-3-one-1,1-dioxide, abbreviated as b) and tetraethyl disulfide (tetraethyl disulfide, abbreviated as t), etc. the structural formula is shown in Figure 1. However, the residual vulcanizing agent in the antisense nucleic acid has side effects on the antisense drug itself and is also toxic to organisms. Therefore, it is necessary to determine their residues in the quality control of antisense oligonucleotide drugs

1 experimental part

1.1 instrument and reagent

hp 5890 gas chromatograph, equipped with FID detector, HP 3394 automatic integrator, HP 5971 high-performance desktop gas chromatography mass spectrometer, equipped with willy138 library, zabspec high-resolution magnetic mass spectrometer, PE 235 high performance liquid chromatograph

standard B and T were purchased from sigma (purity greater than 99%), mercaptoethanol was FLUKA, and other reagents were chromatographic pure

1.2 analysis conditions

gas chromatography conditions: hp-1 nonpolar column (30M γ TM0.52mm γ TM 0.32 μ m)； The column temperature adopts programmed heating: the initial temperature is 150 ℃, which is maintained for 3 minutes, and the temperature is increased to 250 ℃ at 10 ℃/minute, which is maintained for 10 minutes; Injector temperature 250 ℃; Detector temperature 270; The carrier gas is high-purity nitrogen, and the flow rate is 1.2ml/min; Split ratio 15:1; Split injection, injection volume is 1.0 μ L。

gas chromatography-mass spectrometry conditions: hp-5ta column (30M γ TM0.25mm γ TM 0.25 μ m)； The column temperature is programmed to rise: the initial temperature is 80 ℃, rise to 250 ℃ at 15 ℃/min, and maintain for 10 minutes; Injector temperature 250 ℃; Detector temperature 280 ℃; The carrier gas is high-purity helium (purity 99.999%), and the flow rate is 32cm/min; Column head pressure 11.5psi; Split injection, injection volume 1.0 μ Precautions for the use of L projector: the ionization mode is electron bombardment (EI), the interface temperature is 170 ℃, the transmission line temperature is 250 ℃, the electron energy is 70ev, the electron multiplier voltage is 1.3ev, and the solvent delay is 3 minutes

HPLC conditions: reversed phase chromatographic column (250mm γ Tm4.6mm, bio rad company), the mobile phase is a solution of V (methanol): V (water) = 70:30, and the flow rate is 1.0ml/min; Detection wavelength: 220nm; Column temperature 45 ℃, injection volume 50 μ L。

1.3 preparation of standard solution

accurately weigh 11.0mg B of span lang=en-us and put it into a 10ml volumetric flask. Fix the volume with benzene, prepare a standard stock solution of 1.10mg/ml, and dilute it step by step to the required concentration. Similarly, prepare t into a standard stock solution of 1.17mg/ml

1.4 chemical derivatization

take 0.5ml of standard stock solution and add 0.5 μ M NH4HCO3 buffer, and then add mercaptoethanol 10 with a concentration of 1.114g/ml μ 50. React in 55 ℃ water bath. Sampling every hour 150 μ 50. Immediately cool it to -20 ℃ for storage, and take a total of 6 samples. The obtained samples were detected by reversed-phase HPLC and magnetic mass spectrometry. The results showed that the derivatization was complete in 4h, and the best derivatization effect was when the mass fraction ratio of the test drug to mercaptoethanol was 2:1

2 results and discussion

2.1 optimization of gas chromatographic conditions

2.1.1 selection of solvents

carbon disulfide, carbon trichloride, acetone, carbon dichloride, tetrahydrofuran, acetonitrile and benzene are used as solvents respectively. The samples have good solubility in the above solvents, but except acetonitrile and benzene, there are solvent tailing phenomenon and solvent infiltration on the gas chromatogram, so acetonitrile and benzene are used as solvents. In terms of peak shape and repeatability of determination, benzene is more effective than acetonitrile. Therefore, benzene is selected as solvent in the experiment

2.1.2 selection of chromatographic column

three kinds of gas chromatographic columns, namely, polar column hp-innowax, medium polar column bp-225 and non-polar column hp-1, are selected for the experiment, which can be detected. However, using the first two chromatographic columns, the chromatographic peak tailing is serious, which will affect the precision and linear range of the analytical method. This deficiency can be overcome by using the latter chromatographic column hp-1

2.1.3 selection of column temperature

constant temperature and programmed temperature rise were investigated. In terms of resolution and minimum detection limit, programmed temperature rise was better than constant temperature. After repeated experiments, different heating methods were compared to establish the best heating program. At the same time, when selecting the initial column temperature, we should consider not only shortening the analysis cycle, but also considering the sensitivity of the tested object. When the temperature is 100 ℃ ~150 ℃, the peak height of PolyOne carbon fiber composite per unit injection volume helps Corvette C7's new journey basically unchanged, but when the value is greater than 150 ℃, the chromatographic peak is significantly broadened and the peak height is significantly reduced. Therefore, it is appropriate to choose 150 ℃ as the initial column temperature

202 determination of gas chromatography-mass spectrometry

perform gc/ms full scan analysis on the derivatized samples of B and t respectively. See Fig. 2 and Fig. 3 for the mass spectra. According to figure 2 and figure 3, the base peaks of B and T are 168 and 116 respectively. The ions of m/z 168 and m/z 116 were used as characteristic targets to monitor the ions, and the retention time and the retrieval results of wiley138 library were used to further confirm the samples to ensure the accuracy and reliability of the results

2.3 detection limit and linear range

b and t standard chromatograms are shown in Figure 4 and figure 5. In order to determine their minimum detection limit and linear range, the standard solutions with different concentrations were analyzed, and the chromatographic peak areas and peak heights with different injection quantities were obtained. Taking the chromatographic peak height as twice the baseline noise height as the standard to calculate the minimum detection limit, the minimum detection limit of B is 50mg/ml, and the minimum detection limit of T is 100mg/ml

the linear regression equation of the two vulcanizing agents is as follows:

b:y=14228x-248846, r=0.995

t: y=13158x-343695, r=0.998

where x is the injection volume, y is the chromatographic peak area, and the linear range is 150mg/ml-1100mg/ml

3. Conclusion

consumers have reason to believe that under the increasingly perfect service system and good service attitude, antisense oligonucleotides are a new kind of drugs. The relevant research history is not long, especially the systematic drug quality control standards and test method standards have not been established. Due to the heavy workload of the subject and the multi-step research, the research time is tight for the determination of residual vulcanizing agent in antisense oligonucleotides. In this subject, two kinds of the most commonly used vulcanizing agents are studied, but other vulcanizing agents are not involved, which need to be further studied