High-Performance Thin Layer Chromatography (HPTLC) Application and Advantages

HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in a more improved way. It is also known as flat-bed or planar chromatography as the stationary phase used is flatbed like surface.

It works on the same principle of separation by adsorption as conventional TLC. The mobile phase or solvent flows through the capillary action. The analytes move according to their affinities towards the stationary phase (adsorbent). The component with more affinity towards the stationary phase travels slower and the components with less affinity towards the stationary phase travel faster.

Steps Involved in HPTLC:

Application of HPTLC:

  • Forensic Analysis: A challenge in forensic toxicology is the identification of unknown poisonous substances in intoxication cases. HPTLC offers rapid identification as well as qualitative and quantitative analysis for toxic substances.
  • Herbal Applications: HPTLC fingerprint technology can be used in the identification of botanical materials that are very complex in nature.
  • Food Industry: To evaluate nutrients, beverages, vitamins, and pesticides in fruit, vegetables, and other foodstuffs.
  • Pharmaceutical Industry: Used in post-production quality control. analysis of forced degradation studies, stability testing, and to check the presence of impurities in the drug.
  • Cosmetic Industry: HPTLC technique can be employed for the detection of UV filters used in the formulation of cosmetic products. The identified filter can be further confirmed using Mass Spectrometer.

Advantages of HPTLC:

  • It is a flexible, robust, and cost-effective separation techniques employed in the discovery, development, and analysis of new drugs.
  • More than one analyst can work on the system at a time.
  • Shorter developing and analysis time.
  • No prior treatment of solvents like filtration and degassing is required.
  • Fresh stationary and mobile phase is used for each analysis so no interference or contamination comes in the analytical process.
  • It can be used alone or in combination with other techniques like MS, FTIR, Densitometry.

Summary:

HPTLC is the most flexible, reliable, and cost-efficient separation technique so becomes one of the most widely used methods for analysis in pharmaceutical industries. This technique is also used in clinical chemistry, forensic chemistry, biochemistry, cosmetology, food and drug analysis, environmental analysis, and other areas.

Root Cause Analysis and Tools Used for RCA- Pharmaceuticals

Root cause analysis is a methodology to find the primary cause of problem, by tracking back the actions which lead to the nonconformance(problem) and helps to solve the problem.

Root cause analysis is used to identify the problem. What happened? why it happened? and then to determine, what improvements or changes are required to overcome the problem. How it can be prevented in the future.

Steps involved in Root Cause Analysis(RCA):

Tools used for Root Cause Analysis:

There are different tools that can be used to find the exact cause of the problem. A single tool or combination of tools can be used to find the root cause of the problem. Some of the tools used for root cause analysis are listed below:

Brain Storming: In this technique all the people involved in process like experts, frontline worker and people who are familiar with the problem can be brought together to discuss upon the problem and its probable solution.

5 Whys: 5 why is a simple and powerful tool to find the root cause of the problem. In this technique question “why” is asked repeatedly from the statement of the problem till the time the primary root cause is analyzed.

In most of the cases by the fifth “? why” is asked the root cause to a problem is found. Some time it may take fewer or more than five “whys” to find the issue related to the problem.

Cause and Effect Analysis: Is also called as fishbone diagram as it resembles to the skeleton of fish. In this technique all the possible and probable causes of problems are listed and grouped under 5M & E category. (5 M are: Material, Machine, Measurement, Man & Method and E is Environment). Then by studying the graph the factors which do not contribute to the problem are eliminated.

The list of all possible and probable causes to the problem identified in brain-storming and using 5 whys technique can be used in cause and effect diagram to reach to root cause and solution to problem.

Root cause analysis is used in many industries including pharmaceuticals to find the primary cause of the non conformity or problem and its solution, so that the problem is solved from the root and its occurance in future is prevented or at least minimised.

While an RCA provides necessary feedback on an organization’s operational performance, it also costs it both in resources and time. Therefore organizations may choose to investigate only those events which have a critical impact on products or system

Light intensity for Pharmaceutical Area

Lighting at work is very important to the health and safety of everyone using the workplace. Poor lighting can affect the health of the people working in any industry causing headaches, eye strain, and migraine. Poor lighting can harm the business in the form of errors in work, absenteeism, and reduced staff efficiency and productivity.

Excessive lighting can cause glare. Glare is of two types disability glare and discomfort glare. Disability glare is the one when there is direct interference of light with vision resulting in impairment. In discomfort glare vision is not directly impaired, but it may cause discomfort, annoyance, irritability, or distraction the condition is called discomfort glare.

Lighting Requirements:

There are no regulatory guidelines for the intensity of the lighting required. It only states that “Adequate lighting shall be provided in all areas”. Adequate lighting for a particular workplace or area is decided based on the activity carried out in that particular work area and the intensity of light required to do the particular work.

Lux - Wikipedia
Lux Meter

To meet the lighting requirement, it is necessary to define the term “adequate”. This may be done by defining the amount of light (lux) reaching the working surface for each area involved in the production of pharmaceuticals.

It should be ensured that light intensity do not drop below the defined level by monitoring it on defined frequency.

The standard unit of Light intensity is Lux. 1 lux is equal to the light intensity of the surface one meter away from a signal candle. The lux is measured using Lux meter.

Light intensity should be checked at the operational height in the department about one meter from the ground. To determine final light intensity, the light intensity of a minimum five location are recorded and an average of all readings are calculated.

The ideal light intensity for offices is between 300-500 Lux. It should be about 400 lux in all production areas and above 300 lux in sampling and dispensing booth. In the inspection area, the light intensity is above 500 lux to see things better.

Summary: The intensity of Light plays an important in the workplace. Poor light intensity or too much light intensity can affect the quality of work, work output, and health of the people using the area. The intensity of light required for a particular area needs to be studied and defined.

Climatic Zone and Stability Study Conditions as per ICH Guidelines

The climatic condition changes as we move across the globe, which means that the climatic condition is different in a different part of the world. Now since the stability of pharmaceutical product is influenced by climatic conditions, the stability study of the pharmaceutical product needs to be studied keeping in mind the climatic conditions of the country. As per ICH guidelines for stability studies, the climate of the world is divided into four different zones (Zone I, II, III, IV.). Zone IV is further divided into two, zone IV A and zone IV B.

ICH stability zone:

Stability ZonesClimatic Condition
Zone ITemperate Zone
Zone IIMediterranean /Sub-tropical Zone
Zone IIIHot Dry Zone
Zone IV AHot Humid Tropical Zone
Zone IV BHot/Higher Humidity

As the climatic zones are differentiated based on the temperature and humidity of different part of the world. ICH has defined different stability condition for studies as per the Stability zone. The stability study condition for Drug Product is mentioned below:

Long Term Stability Testing Conditions
Stability ZoneTemperatureHumidityMinimum Duration
Zone I21°C ± 2°C45 %RH ± 5 % RH12 Months
Zone II25°C ± 2°C60 %RH ± 5 % RH12 Months
Zone III30°C ± 2°C35 %RH ± 5 % RH12 Months
Zone IV A30°C ± 2°C65 %RH ± 5 % RH12 Months
Zone IV B30°C ± 2°C75 %RH ± 5 % RH12 Months

Intermediate and Accelerated Stability Testing Condition:

StudyStorage conditionMinimum Duration
Intermediate30°C ± 2°C/65% RH ± 5% RH6 Months
Accelerated40°C ± 2°C/75% RH ± 5% RH6 months

For the Drug Productintended for storage in a refrigerator:

StudyStorage ConditionMinimum Duration
Long Term 5°C ± 3°C12 months
Accelerated25°C ± 2°C/60% RH ± 5% RH6 months

For the Drug Product intended for storage in a freezer:

StudyStorage ConditionMinimum Duration
Long Term-15°C ± 5°C/ No Humidity12 months
Accelerated5°C ± 3°C/No Humidity6 months

A successful stability study establishes the shelf-life of the drug product, the retest period of a drug substance, and appropriate storage conditions. A successful stability study must also ensure that patients receive safe and effective medicine.

System Suitability Test Requirements in Chromatography

System suitability test (SST) is a test to determine the suitability and effectiveness of a chromatographic system prior to use. The performance of any chromatographic system may continuously change during their regular use, which can affect the reliability of the analytical results.

The operation parameters of the whole chromatographic system can be checked with properly selected SST mixtures. System suitability testing limits are the acceptance criteria that must be met prior to the use of sample analysis.

The system suitability testing limit should conform to criteria provided in guidelines by CDER and other pharmacopeial references like USP and ICH. Some of the parameters which can be checked as SST requirements are: 

  • Capacity Factor
  • Resolution
  • Tailing Factor
  • Theoretical Plate Number
  • Signal to Noise ratio
  • Repeatability

Let’s look in to each of the System Suitability parameters :

Capacity Factor: The capacity factor is a measure of, where the peak of interest is located with respect to void volume, i.e., elution time of the non-retained components. Generally, the value of K is > 2.

Resolution: Resolution is an important HPLC performance indicator. It is used to ensure that closely eluting compounds are resolved from each other as they pass through the column in the given condition. Resolution of > 2 between the peak of interest and the closest potential interfering peak is desirable.

Tailing Factor: Tailing factor is a measure of peak symmetry. It is calculated as Tf = (a +b) / 2a, where ‘a’ and ‘b’ are the peak half-widths at 5% of the peak height, a is the front half-width, b is the back. Tailing factor of < 2 is generally recommended.

Theoretical Plate Number(N): The theoretical plate number is a measure of column efficiency. H, or HETP, the height equivalent of a theoretical plate, measures the column efficiency per unit length (L) of the column. peak position, particle size in the column, flow-rate of the mobile phase, column temperature, viscosity of the mobile phase, and molecular weight of the analyte are some of the parameters which can affect Plate number.

Signal to Noise ratio: The signal-to-noise (S/N) ratio is often used to measure the sensitivity of a liquid and gas chromatography instrument. 

Repeatability: The closeness of the agreement between the results of successive measurements carried out under the same conditions of measurement.

Summary: Retention time, pressure, and other criteria may be included as system suitability test parameters. The system suitability parameters may not be the same for all the analytical methods. All the parameters mentioned in this article may not be considered for all analytical methods, the minimum system suitability test parameters requirement changes with the purpose of testing.

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Friability Testing – Friability of uncoated tablets

Friability (the condition of being Friable) testing is a methodology, used to see the physical strength of compressed and uncoated tablets upon exposure to mechanical shock and attrition. In simple words, what quantity of mechanical stress tablets will stand up to throughout their production, distribution, and handling process.

Friability Test Apparatus: Friability test apparatus consists of a drum of a transparent synthetic polymer with polished internal surfaces having minimum static charge. One facet of the drum is removable.

Dimensions: The Internal diameter of the drum is between 283 and 291 mm and a depth between 36 and 40 mm. The tablets fall down with each turn of the drum by a curved projection with an inside radius between 75.5 and 85.5 mm that extends from the middle of the drum to the outer wall. The outer diameter of the central ring is 24.5 mm to 25.5. The drum is attached to the horizontal axis of a device that rotates at 25 ±1 rpm

Test Method:

For tablets with a unit mass equal to or less than 650 mg, take a sample of whole tablets corresponding to 6.5 g. For tablets with a unit mass of more than 650 mg, take a sample of 10 whole tablets.

Dedust the tablet carefully and weigh accurately the tablet sample. Place the tablets in the drum. Rotate the drum 100 times with a speed of 25 rpm, remove the tablets, remove any loose dust from the tablets as before, and accurately weigh.

Calculation:

Friability (%)  = W1 – W2/ W1  X 100

Where,
W1 = Weight of Tablets (Initial / Before Tumbling) &
W2 = Weight of Tablets (After Tumbling or friability)

Limit : Friability (%) = Not More Than 1.0 %

Generally, the test is run once. If cracked, cleaved, or broken tablets are present in the tablet sample after tumbling, the sample fails the test. If the results are doubtful or if the weight loss is greater than the targeted value. The test should be repeated twice and the mean of the three tests should be calculated. A maximum mean weight loss from the three samples of not more than 1.0% is considered acceptable for most products.

If tablet size or shape causes irregular tumbling, the drum base should be adjusted, so that the base forms an angle of about 10° with the bench top and the tablets do not bind together when lying next to each other, which prevents them from falling freely.

An appropriate humidity­ controlled environment is required for testing of hygroscopic tablets.

Summary:

  • Friability is defined as the % of weight loss by tablets due to mechanical action during the test.
  • Rotate the drum 100 times with a speed of 25 rpm.
  • For tablets with a unit mass equal to or less than 650 mg, take a sample of whole tablets corresponding to 6.5 g.
  • For tablets with a unit mass of more than 650 mg, take a sample of 10 whole tablets.
  • Limit: Friability % : Not more than: 1.0%

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