Pharmaceutical Analysis

(Code:1002341)

 

Guideline

I. Course Introduction

112 classes comprise 72 for lecturing, 40 for lab practice.

7.0 credits; 5th semester

II. Lecturing section

Lecturing and Research Section, Department of Pharmaceutical Analysis, School of Pharmacy

III. Attribution

Pharmaceutical analysis is a subject builting on the experiment. The function of this course is to teach the strudents to buid the view of comprehensive quality control of drugs, to make the students know the methods and technologies of pharmaceutical analysis, further to let the students do test works on the drug research, drug manufacture, drug supply, drug clincial usuage and drug market supervision, and to let the students have the ability of doing creative research and soving the problems of drug quality.

IV. Requirements

The course is on the basis of chemistry, biology and pharmacy. By learning this course, the students is required to know the rules of drug quality management and the function of pharmaceutical analysis; to know the content of drug quality research and how to make the quality standard for a given drug; to know the content of pharmacopoeia and the relative applications in drug analysis; to know the common rules and methods of identification, impurity test and quantitative analysis of drugs; to know the quality rules and analytical characteristic of typical drugs; to know the modern analytical technology and development on drug quality research.

V. Compulsory subject

 Analytical Chemistry; Organic Chemistry; Biochemistry, Pharmaceutical Chemistry, etc.

Contents

Lecturing

 Class Discussion

Self-study

Lab

Chapter 1

Control of the Quality of Analytical Methods

2

 

 

 

  Chapter 2

Extraction Methods in Pharmaceutical Analysis

6

 

 

4

Chapter 3

Physical and Chemical Properties of Drug Molecules

5

 

 

4

Chapter 4

Titrimetric and Chemical Analysis Methods

5

 

 

4

Chapter 5

Chromatography theory

5

 

 

 

Chapter 6

Thin-layer Chromatography

2

 

 

 

Chapter 7

Gas Chromatography

4

 

 

8

Chapter 8

High-performance Liquid Chromatography

4

 

 

8

Chapter 9

Nuclear Magnetic Resonance Spectroscopy

3

 

 

 

Chapter 10

Mass Spectrometry

6

 

 

 

Chapter 11

Ultraviolet and Visible Spectroscopy

4

 

 

8

Chapter 12

Infrared Spectrophotometry

4

 

 

 

Chapter 13

Atomic Spectrophotometry

2

 

 

 

Chapter 14

Molecular Emission Spectroscopy

6

 

 

4

Chapter 15

High-performance Capillary Electrophoresis

6

 

 

 

Chapter 16

Additional Information about Pharmaceutical Analysis

8

 

 

 

Total

72

 

 

40

 VI. References

  1. Textbook

David G. Watson, Pharmaceutical Analysis, 3th ed. Elsevier Publishing.

Hang Taijun, Pharmaceutical Analysis, 7rd, ed. People's Medical Publishing House.

Analytical Chemistry, Dalian University of Technology press.

  1. Journal

Analytical Chemistry, ACS publishing.

Analytical Chimica Acta, Talanta, Journal of Chromtography A,  Journal of Chromtography B, Elsevier publishing.

3.  Website

http://pubs.acs.org/

http://www.sciencedirect.com/science/journals

http://pubs.rsc.org/

http://onlinelibrary.wiley.com/

http://link.springer.com/

VII. Course organization

10% attendence; 10% homework; 30% experiment; 50% final examine


 

Chapter I Control of the Quality of Analytical Methods

  1. Objective and requirements

To master the properties and the task of pharmaceutical analysis;

To understand the role of pharmaceutical analysis in the quality control of a drug.

  1. key points

The attribution and the task of pharmaceutical analysis;

  1. Lecturing contents and important points

    1. To introduce the questions pharmaceutical analysis methods are used to answer.

    2. To introduce the guidelines of International Conference on Harmonisation (ICH).

IV. Definitions

V. Study questions

    1. What is the questions pharmaceutical analysis methods are used to answer?

    2. What are the guidelines of International Conference on Harmonisation (ICH)?

 

Chapter 2 Extraction Methods in Pharmaceutical Analysis

  1. Objective and requirements

To master the principles of extraction;

   To know the solvent extraction method;

   To know solid-phase extraction.

II. Key points

The principles of extractions, including solvent extraction and solid-phase extraction and know their applications in pharmaceutical analysis.

III. Lecturing contents and important points

   1. The principles of extraction;

   2.The solvent extraction method and the types;

   3. The solid-phase extraction and the types;

   4. The application of the extraction methods;

   5. The strengths of the extraction methods;

    6. The limitations of the extraction methods.

IV. Definitions

   Solvent extraction; solid-phase extraction; mcrodialysis extraction

V. Study questions

   1. What are the principles of extractions?

   2. What are the types of solvent extraction?

   3. What is the types of solid-phase extraction?

 

Chapter 3 Physical and Chemical Properties of Drug Molecules

  1. Objective and requirements

    To master the roles of physical and chemical properties of drug molecules in the design of analytical methods;

    To know the physical and chemical properties of drug molecules.

  1. Key points

   The physical and chemical properties of drug molecules

  1. Lecturing contents and important points

1. Calculation of pH value of aqueous solutions of strong and waek acids and bases;

2. Acidic and basic strength and pKa;

3. Henderson-hasselbalch equation;

4. Ionisation of drug molecules;

5. Buffers;

6. Salt hydrolysis;

7.Activity, ionic strength and electric constant;

8. Partition coefficient;

9. Drug stability;

10. Stereochemistry of drugs;

11. Measurement of optical rotation;

12. Profiles of physico-chemical properties of some drug molecules.

  1. Definitions

Partition coefficient; optical rotation

  1. Study questions
  1. How to calculate the pH of a given solution with the known concentration and activity?

   2. How to predict the percentage of ionisation of the functional groups specified in the given drugs at pH value?

   3. How to calculate the percentage of extraction of given compounds under the conditions specified?

 

Chapter 4 Titrimetric and Chemical Analysis Methods

  1. Objective and requirements

To master the principles of titrimetric and chemical analysis methods;

    To understand the applications of titrimetric and chemical analysis methods;

    To know the advantages and limitations of titrimetric and chemical analysis methods.

II. Key points

    The principles of titrimetric and chemical analysis methods and the relative applications

III. Lecturing contents and important points

  1. Introduction;
  2. Instrumentation and reagents;
  3. Direct acid/base titrations in the aqueous phase;
  4. Titrations of the salts of weak bases in mixed aqueous/non-aqueous media;
  5. Indirect titrations in the aqueous phase;
  6. Non-aqueous titrations;
  7. Argentimetric titrations;
  8. Compleximetric titrations;
  9. Redox titration;
  10. Iodometric titrations;
  11. Ion pair titrations;
  12. Diazotisation titrations;
  13. Potentiometric titrations;
  14. Karl Fischer titration;
  15. Automation of wet chemical methods;
  16. Applications of FIA in pharmaceutical analysis;
  17. Additional problems.

IV. Definitions

Titration; non-aqueous titrations; redox titration; flow injection analysis

  1. Study questions
  1. What are the principles of titrations?
  2. What are the advantages of titrations? and the limitations?
  3. Calculation

 

Chapter 5 Chromatography Theory

I. Objective and requirements

To master the chromatography theory

 II. Key points

   The chromatography theory.

III. Lecturing contents and important points

  1. Introduction;
  2. Void volume and capacity factor;
  3. Calculation of column efficiency;
  4. Origins of band broadening in HPLC (Van Deemter equation in liquid; Chromatography;Van Deemter equation in gas chromatography);
  5. Parameters used in evaluating column performance (resolution; peak asymmetry);
  6. Data acquisition;
  7. Report generation.

IV. Definitions

Column efficiency; capacity factor; band broadening; resolution; peak asymmetry

  1. Study questions
  1. How to calculate the column efficiency?
  2. How to evaluate the separation system?

 

Chapter 6 Thin-layer Chromatography (TLC)

  1. Objective and requirements

To master the mechanism of TLC;

To master the compartments of TLC instrumentation;

To understand the parameters of TLC affecting the drug analysis;

To understand the applications of TLC in pharmaceutical analysis;

To know the latest technology of of TLC.

II. Key points

The mechanism of TLC; the compartments of TLC;  the parameters of TLC affecting the drug analysis; the applications of TLC in pharmaceutical analysis

III. Lecturing contents and important points

  1. Introduction;
  2. Instrumentation;
  3. TLC chromatogram;
  4. Stationary phases;
  5. Elutropic series and mobile phases;
  6. Modification of TLC adsorbant;
  7. Detection of coumpounds on TLC plates following development;
  8. Applications of TLC analysis (Qualitative identity tests; Limit tests; Tests in which known and unknown standards are used);
  9. High-performance TLC (HPTLC).

IV. Definitions

   TLC, High-performance TLC

V. Study questions

  1. What is the mechanism of TLC?
  2. What are the compartments of TLC instrumentation?
  3. Predict the orders of different compounds in TLC system based on the given conditions?
  4. Indicate the applications of TLC in pharmaceutical analysis?
  5. Indicate parameters of TLC in pharmaceutical analysis for a specified drug?

 

Chapter 7 Gas Chromatography (GC)

  1. Objective and requirements

To master the mechanism of GC;

To master the compartments of GC instrumentation;

To understand the applications of GC in pharmaceutical analysis;

To know the latest applications of GC coupled with new sample preparation.

II. Key points

The mechanism of GC; the compartments of GC instrumentation;  the parameters of GC affecting the drug analysis; the applications of GC in pharmaceutical analysis

III. Lecturing contents and important points

  1. Introduction;
  2. Instrumentation;
  3. Selectivity of liquid stationary phases;
  4. Use of derivatisation in gas chromatography;
  5. Summary of parameters governing capillary gas chromatography performance;
  6. Gas chromatography detectors;
  7. Applications of gas chromatography in quantitative analysis;
  8. Determination of manufacturing and degradation residues by gas chromatography;
  9. Determination of residual solvents;
  10. Solid-phase microextraction (SPME);
  11. Applications of gas chromatography in bioanalysis;
  12. Additional problems.

IV. Definitions

   GC, residual solvents, solid-phase microextraction

V. Study questions

  1. What is the mechanism of gas chromatography?
  2. What are the compartments of gas chromatography instrumentation?
  3. Predict the orders of different compounds in GC system based on the given conditions?
  4. Indicate the applications of GC in pharmaceutical analysis?
  5. Indicate parameters of GC in pharmaceutical analysis for a specified drug?

 

Chapter 8 High-performance Liquid Chromatography (HPLC)

  1. Objective and requirements

   To master the mechanism of HPLC;

   To master the compartments of HPLC instrumentation;

   To understand the parameters of HPLC affecting the drug analysis;

   To understand the applications of HPLC in pharmaceutical analysis;

   To know the latest applications of HPLC in pharmaceutical analysis.

II. Key points

   The mechanism of HPLC; the compartments of HPLC instrumentation;  the parameters of HPLC affecting the drug analysis; the applications of HPLC in pharmaceutical analysis.

III. Lecturing contents and important points

   1. Introduction;

   2. Instrumentation;

   3. Stationary and mobile phases;

   4.Structural factors which govern rate of elution of compounds from HPLC columns;

   5.More advanced consideration of solvent selectivity in reverse-phase chromatography;

   6. Effect of temperature on HPLC;

   7. Summary of stationary phases used in HPLC;

   8.A more advanced consideration of reverse-phase stationary phases;

   9.Summary of detectors used in HPLC;

   10.Performance of a diode array detectors;

   11.Applications of HPLC to the quantitative analysis of drugs in formulations;

   12.Assays involving more specialised HPLC techniques;

   13. Additional problems.

IV. Definitions

   HPLC; resolution; reverse-phase stationary phase

V. Study questions

  1. What is the mechanism of HPLC?
  2. What are the compartments of HPLC instrumentation?
  3. Predict the orders of different compounds in HPLC system based on the given conditions?
  4. Indicate the applications of HPLC in pharmaceutical analysis?
  5. Indicate parameters of HPLC in pharmaceutical analysis for a specified drug?

 

Chapter 9 Nuclear Magnetic Resonance (NMR) Spectroscopy

  1. Objective and requirements

To master the principles of NMR;

To master the spectra of NMR and know how to predict the structure confirmation of compound;

To understand the applications of NMR in pharmaceutical analysis.

  1. Key points

The principles of NMR; the spectra of NMR; the applications of NMR in pharmaceutical analysis.

  1. Lecturing contents and important points
  1. Introduction;
  2. Instrumentation;
  3. Proton (1H) NMR;
  4. Application of NMR to structure confirmation in some drug molecules;
  5. Carbon NMR;
  6. Two-dimensional NMR spectra;
  7. Application of NMR to quantitaive analysis;
  8. Other specialised applications of NMR.

IV. Definitions

NMR; Carbon NMR; Two-dimensional NMR

V. Study questions

  1. What is the principles of NMR?
  2. What are the compartments of NMR instrumentation?
  3. Predict the possible structure of the molecule based on the given spectra of NMR?
  4. Indicate the applications of NMR in pharmaceutical analysis?
  5. Indicate further development of NMR in pharmaceutical analysis?

 

 Chapter 10 Mass Spectrometry (MS)

  1. Objective and requirements

To master the principles of MS;

To master the spectra of MS and know how to predict the structure confirmation of compound;

To understand the applications of MS in pharmaceutical analysis;

To understand the strengths and limitations of MS in applications;

To know the latest hyphenated technology of MS and the relative applications.

  1. Key points

The principles of MS; the applications of MS in pharmaceutical analysis;

the strengths and limitations of MS; the hyphenated technology of MS.

  1. Lecturing contents and important points
    1. Introduction;
    2. Ion generation;
    3. Other ionisation methods;
    4. Ion separation techniques;
    5. A more detailed consideration of mass spectra;
    6. Molecular fragmentation patterns;
    7. Gas Chromatography-mass spectrometry (GC-MS);
    8. Applications of GC-MS;
    9. Tandem mass spectrometry;
    10. High-resolution mass spectrometry;
    11. Mass spectrometry of proteins;
    12. Mass spectrometry in drug discovery.

IV. Definitions

MS; Tandem mass spectrometry

  1. Study questions
  1. what are the ion generation in MS?
  2. What are the ion separation techniques in MS?
  3. What are the principles of MS?
  4. What are the strengths and limitations of MS in applications?
  5. Indicate the applications of MS related techniques in pharmaceutical analysis?
  6. Deduce the possible structure of the molecule based on a given spectrum of MS?

 

Chapter 11 Ultraviolet and Visible Spectroscopy (UV-vis)

  1. Objective and requirements

To master the principles of UV-vis;

To master the application of UV-vis in pharmaceutical analysis;

To understand the strengths and limitations of UV-vis in applications.

  1. Key points

   The principles of UV-vis; Beer-Lambert Law; pharmaceutical quantitative analysis.

  1. Lecturing contents and important points
  1. Introduction;
  2. Factors governing absorption of radiation in the UV/visible region;
  3. Beer-Lambert Law;
  4. Instrumentation;
  5. Diode array instruments;
  6. Instrument calibration;
  7. UV spectra of some representative drug molecules;
  8. Use of UV/visible spectrophotometry to determine pKa values;
  9. Applications of UV/visible spectroscopy to pharmaceutical quantitative analysis;
  10. Difference spectrophotometry;
  11. Derivative spectra;
  12. Applications of UV/visible spectroscopy in preformulation and formulation;
  13. Additional problems.
  1. Definitions

UV-vis; Beer-Lambert Law

  1. Study questions

    1. What are the principles of UV-vis?

    2. Analyse the relationship of the absorpion of spectra and the structure of molecule?

    3. Calculate the concentrations of drugs based on Beer-Lambert

  

Chapter 12 Infrared (IR) Spectrophotometry

  1. Objective and requirements

To master the principles of IR;

To master the application of IR in pharmaceutical analysis;

To understand the strengths and limitations of IR in applications;

To know the sample preparation in IR for different states of targets;

To know the technique of NIRA and its applications in pharmaceutical analysis.

  1. Key points

   The principles of IR; pharmaceutical qualitative analysis.

  1. Lecturing contents and important points
  1. Introduction;
  2. Factors governing intensity and energy level of absorption in IR spectra;
  3. Instrumentation;
  4. Sample preparation;
  5. Application of IR spectrophotometry in structure elucidation;
  6. Examples of IR spectra of drug molecules;
  7. IR spectrophotometry as a fingerprint technique;
  8. IR spectrophotometry as a method for identifying polymorphs;
  9. Near-infrared analysis (NIRA);
  10. Examples of NIRA applications;
  11. Additional problems.
  1. Definitions

IR; NIRA

  1. Study questions

    1. What are the principles of IR?

    2. What are the sample preparations for different states of smaples in IR?

    3. Find the characteristic absorptions of the function groups for a given molecule in IR spectra

 

Chapter 13 Atomic Spectrophotometry

  1. Objective and requirements

    To master the principles of atomic emission spectrophotometry (AES) and atomic absorption spectrophotometry (AAS);

To know the applications of AES and AAS in pharmaceutical analysis;

To know the strengths and limitations of AES and AAS in applications.

  1. Key points

    The principles of AES and AAS; The applications of AES and AAS in pharmaceutical analysis.

  1. Lecturing contents and important points
  1. The principles of AES and AAS;
  2. The instrumentations of AES and AAS;
  3. The applications of AES and AAS in pharmaceutial analysis.

IV.  Definitions

    AAS, AES, ICP, standard addition method

V.   Study questions

  1. What are the principles of AAS and AES?
  2. Indicate the applications of AAS and AES in pharmaceutical analysis?

 

          Chapter 14 Molecular Emission Spectroscopy

  1. Objective and requirements

   To master the principles of fluorescence spectrophotometry;

To master the applications of fluorescence spectrophotometry in pharmaceutical analysis;

To know the principles of Raman spectroscopy;

To know the applications of Raman spectroscopy in pharmaceutical analysis.

  1. Key points

The principles of fluorescence spectrophotometry and its applications in pharmaceutical analysis;

The principles of Raman spectroscopy and its applications in pharmaceutical analysis;

  1. Lecturing contents and important points
  1. Fluorescence spectrophotometry (the principles; instrumentation and applications);
  2. Raman spectroscopy (the principles; instrumentation and applications).
  1. Definitions

Fluorescence spectrophotometry; Raman spectroscopy

  1. Study questions
  1. What are the principles of fuorescence spectrophotometry?
  2. What are the strengths and limitations of fluorescence spectrophotometry in pharmaceutical analysis?

 

Chapter 15 High-performance Capillary Electrophoresis (HPCE)

  1. Objective and requirements

   To master the principles of capillary electrophoresis (CE);

To master the applications of CE in pharmaceutical analysis;

To know the various modes of CE in applications;

To know the strengths and limitations of CE.

  1. Key points

   The principles of CE; the applications of CE in pharmaceutical analysis; the various modes of CE in applications

  1. Lecturing contents and important points
  1. Introduction (electrophoresis; EOF; Migration in CE);
  2. Instrumentation;
  3. Control of separation (Migration time; dispersion);
  4. Applications of CE in pharmaceutical analysis;
  5. Use of additives in the running buffer;
  6. Various modes of CE in applications;
  7. Additional problems.
  1. Definitions 

Electrophoresis; EOF

  1. Study questions

1. What are the principles of CE?

2.Describe the formation of EOF in CE system?

3. Predicting the elution order of analytes in a given CE system?

 

Chapter 16 Additional Information about Pharmaceutical Analysis

  1. Objective and requirements

   To know the pharmacopoeia in the world;

   To know the contents of pharmacopoeia;

To know the contents of quality control for a drug substance and a drug product;

To know the general process to do a quality control research;

To know the identification test of drug and know the relative techniques;

To know the impurity test of drug and know the relative techniques;

To know the quantitative analysis of drug and know the relative techniques.

  1. Key points

   Pharmacopoeia; identification test; impurity test; quantitative analysis

  1. Lecturing contents and important points

1. The pharmacopoeia in the world;

2. The contents of pharmacopoeia;

3. The contents of quality control for a drug substance and a drug product;

4. The general process to do a quality control research;

5. The identifying test of drug and know the relative techniques;

6.The impurity test of drug and know the relative techniques;

7.The quantitative analysis of drug and know the relative techniques;

  1. Definitions 

Pharmacopoeia; identification test; impurity test; quantitative analysis

  1. Study questions

1.Try to build a qulity control process for a given drug?


 

Experiments For Pharmaceutical Analysis

Experiment 1 Impurities test of glucose

Objective

  1. To master the principle of identification of glucose.
  2. To master the general impurities items and the calculation of impurity limitation.
  3. To master the principles of testing general impurities and their methods.

Principle

1. Identification test

Aldehyde or ketone group has reductive, and can reduce divalent copper into cuprous oxide in alkaline cupric tartrate TS (Fehling test solution). This assay has been applied to identify anhydrous glucose, glucose injection, glucose and sodium chloride injection.

2. Acidity

It is to check the impurities with acidity or alkalinity using the method prescribed in the pharmacopoeia. New boiled water when cool to room temperature is required as the solvent. Organic solvents, such as neutral ethanol, can be used to dissolve the drug that is insoluble in water. Commonly used methods are acid-base titration, indicator method and pH assays.

3. Chloride

Chloride can react with silver nitrate in a nitric acid solution, generating silver chloride precipitate and showing white turbid. The drug’s chloride test limit is determined by comparing the degree of turbidity of silver chloride between the drug and the standard sodium chloride solution, which are treated in the same process under the same conditions.

Cl- + Ag+ → AgCl↓(white turbid)

4. Iron

Ferric salts in nitric acid solution can form a red-brown soluble ferric thiocyanate complex ion with thiocyanate, and its test limit is obtained by colorimetric method with a reference iron solution after treated with the same method.

Fe3+ + 3SCN- → Fe(SCN)3(red-brown)

Equipment and Chemicals

1. Alkaline cupric tartrate TS

(1) Dissolve 6.93g of crystalline cupric sulfate in water to make 100 mL; (2) Dissolve 34.6g of crystalline potassium solution tartrate and 10 g of sodium hydroxide in water to make 100 mL; (3) Mix solution (1) with solution (2) before use.

2. Phenolphthalein indicator solution (IS)

Dissolve 1g of phenolphthalein in 100ml of ethanol. Colour changes from colourless to red (pH 8.3-10.0)

3. Blue litmus TP

4. Sodium hydroxide (0.02mol/L) VS

5、Nitric acid dilute TS

Dilute 105 ml of nitric acid in water to make 1000 mL. It contains 9.5%-10.5% of HNO3.

6. Hydrochloric acid dilute TS

Dilute 234 mL of hydrochloric acid with water to 1000 mL. It contains 9.5%-10.5% of HCl.

7. Silver nitrate VS

Dissolve 17.5 g of silver nitrate in water to make 1000 mL and mix well. A process of standardization is required to obtain the exact concentration.

8. Sodium chloride standard solution

dissolve 0.165 g of sodium chloride in water in a 1000 mL volumetric flask, and dilute to the volume, mix well (stock solution). transfer 10 mL of the stock solution, accurately measured, into a 100 mL volumetric flask immediately before use, dilute with water to the volume and mix well (each mL is equivalent to 10 μg of Cl)

9. Potassium sulfate standard solution

Dissolve 0.181 g of potassium sulfate in water in a 1000 mL volumetric flask, and dilute to the volume, mix well (each ml is equivalent to 100 μg of SO4).

10. Iodine TS

Dissolve 13.0g of iodine and 36g of potassium iodine in 50 mL of water, add 3 drops of hydrochloric acid and dilute with water to 1000 mL, mix well and filter with a sintered glass filter.

11. Ammonium thiocyanate solution (30→100)

Dissolve 30 g of ammonium thiocyanate in water to make 100 mL.

12. Iron standard solution

Dissolve 0.863 g of ferric ammonium sulfate FeNH4(SO4)2·12H2O, accurately weighed, in water in a 1000 mL volumetric flask, add 2.5 mL of sulfuric acid, dilute with water to volume and mix well. This is the stock solution. Transfer 10 mL of stock solution, accurately measured, to a 100 mL volumetric flask immediately before use, add water to volume and mix well (each mL is equivalent to 10 μg Fe).

Procedure

1. Identification Test

Dissolve about 0.2 g in 5 mL of water, add dropwise hot alkaline cupric tartrate TS; a red precipitate of cuprous oxide is produced.

2. Acidity

Dissolve 2.0 g in 20 mL of water, add 3 drops of phenolphthalein IS and 0.20 mL of sodium hydroxide (0.02mol/L) VS; a pink colour is produced.

3. Chloride

Test preparation: weigh 0.60 g of the substance, dissolve it in about 25 mL of water (if the solution is alkaline, neutralize with nitric acid dropwise, using blue litmus as test paper). Add 10 mL of dilute nitric acid and filter if necessary, transfer the solution to a 50 ml Nessler cylinder, add water to produce 40 mL and mix well.

Reference preparation: transfer 6.0 mL of sodium chloride standard solution to a 50 mL Nessler cylinder, add 10 mL of dilute nitric acid and sulfficient water to produce 40 mL, mix well.

Procedure: to each of the Nessler cylinders above add 1.0 mL of silver nitrate TS, dilute with water to 50 mL and mix well. Allow to stand in the dark for 5 min, compare the opalescence produced by viewing down the vertical axis of the cylinder against a black background.

4. Sulfites and soluble starch

Dissolve 1.0 g in 10 mL of water, add 1 drop of iodine TS; a yellow colour is produced.

5. Sulfate

Test preparation: weigh 2.0 g of the substance being examined as prescribed under individual monographs, dissolve it in about 40 mL of water, neutralize the solution with hydrochloride acid and filter if necessary. Transfer the solution to a 50 mL Nessler cylinder, add 2 mL of diluted hydrochloric acid and mix well.

Reference preparation: Transfer 2.0 mL of potassium sulfate standard solution as described under individual monographs to a 50 mL Nessler cylinder, dilute with water to about 40 mL, add 2 mL diluted hydrochloric acid and mix well.

Procedure: To each of the Nessler cylinders described above add 5 mL of 25% barium chloride solution, dilute with water to 50 mL and mix well, allow to stand for 10 min and compare the opalescence produced by viewing down the vertical axis of the cylinder against a black background. Any opalescence produced is not more pronounced than that of the reference using 2.0 mL of potassium sulfate standard solution (0.01%).

If the test preparation is coloured, unless otherwise specified, place two aliquots of the test preparation in Nessler cylinders separately, to one cylinder add 5 mL of 25% barium chloride solution, mix well and allow to stand for 10 min, filter the content of the cylinder repeatedly until the filtrate is perfectly clear, then add the prescribed volume of potassium sulfate standard solution to the filtrate and use it as the reference preparation. To the other cylinder add 5 mL of 25% barium chloride solution, use it as the test preparation. Dilute the test preparation and the reference preparation with water to 50 mL, mix well and allow to stand for 10 minutes, compare the opalescence as described above.

6. Iron

Dissolve 2.0 g in 20 mL of water, add 3 drops of nitric acid, boil gently for 5 minutes. Allow to cool, dilute to 45 mL with water, add 3 mL of ammonium thiocyanate solution (30→100) and mix well. Any colour produced is not more intense than that of a reference solution prepared in the same manner of iron standard solution (0.001%).

Notes

1. Proper use of colorimetric tube: select the paired Nessler colorimetric tube; remove the dirt by cleaning fluid, then clean with water. Rotary shaking method can mix the liquid well.

2. The correct selection of measuring tools: select the appropriate equipment with capacity based on the general tolerance of ± 10% of the requirements for the check test and the amount of drug and reagent.

3. Parallel operation - standard experiments must be carried out simultaneously with the sample, add consistent amount of reagents. When observed, the degree of illumination of the two tubes should be consistent, make light shine into the front, set on a white background when colorimetric, set on the black background when compare turbidity, top to bottom observation.

4. Note the correct use of scale straw and observation.

5. Calculation of the limit of impurity: impurity limit % = ((V standard × C standard) / W sample) × 100%.

Where V standard is the volume of the standard liquid, C standard is the standard concentration, W is sample volume.

Discussion

1. Turbidimetric method test is to place the two tubes on a black background, and compare the opalescence produced by viewing down the vertical axis of the cylinder against a black background.

Colorimetric method test is to place the two tubes on or against a white background, and view from one side or top-down. The used colorimetric tubes should have consistent scale height.

2. To items of sulfite and soluble starch, iodine test solution fade at the presence of sulfites, and iodine test solution becomes blue at the presence of soluble starch.

 

 

 

 

 


 

Determination of organic solvent residues in drug substance by capillary gas chromatography

Objective

  1. Grasp quantitative method for internal standardization comparison.
  2. Grasp the GC method for determination of residues of organic solvent in drug substance
  3. Grasp the operation for GC system.

Principles

Internal standardization comparison is generally used for quantitative analysis in GC because corrective factors of many organic compounds are unknown. First, standard solution of known concentration is prepared, to which internal standard compound is added. Then sample solution is prepared, which contains internal standard compound of same concentration. After injection these solutions are separated by GC system. The concentration of analyte in sample solution can be calculated according to the following formula:

Where Ci% is the concentration of analyte, Ai and As are the peak area of analyte and internal standard compound, respectively.

Equipment and chemicals

Equipment

Agilent 7820A gas chromatographic system, microsyringe (10μL), volumetric flask (100ml, 10 ml)

Chemicals

Methanol, acetone, n-propanol (inner standard), drug substance: dexamethasone sodium phosphate

Procedure

  1. preparation of solutions
  1. Standard solution  Dissolve 10 μL (7.9 mg) of methanol and 100 μL (79 mg) of acetone into 100 mL volumetric flask, then add 20.0 mL of 0.1% (v/v) n-propanol solution, dilute with water to volume.
  2. Sample solution  Accurately dissolve 0.16 g of dexamethasone sodium phosphate by water in 10 mL volumetric flask, then add 2.0 mL of 0.1% (v/v) n-propanol solution, dilute with water to volume.
  1. Chromatographic conditions

OV-17 capillary column (Agilent, 30m×0.32mm×0.5μm), flame-ionization detector (FID), temperature of injector: 200℃, temperature of detector: 250 ℃, column ovens: 60 ℃, 10 min; split ratio: 50:1, carrier gas, N2, 1.0 ml/min.

  1. Injection

Inject 1μL of standard solution and sample solution respectively under the above conditions.

  1. Calculations

Fill in the blanks in the following table and calculate the concentration of acetone in sample according to the above formula:

 

Name

bp(℃)

tR

A

Ai/As

(Ci%)sample

Standard solution

Acetone

Methanol

n-propanol

56

64.7

97

 

 

 

 

Sample solution

Acetone

Methanol

n-propanol

56

64.7

97

 

 

 

 

The result complies with the related requirements: The content of acetone is not more than 5.0% (g/g) and no methanol peak is detected.

Notes

  1. Don’t pull the valve out of microsyringe’s tube when microsyringe is used.
  2. Rinse the microsyringe over ten times with ethanol to prevent filling after it is used to suck sample solution.

Questions

  1. What is the flow ratio of H2 to N2 to air generally when FID is used?
  2. When is internal standardization comparison used? Must inject volume be accurate when internal standardization is used?
  3. What occurs to the result in this experiment if the flow rate of carrier gas varies a little?