Review on assessment of different analytical techniques for emulsion made for treatment of Jaundice

Jaundice, also known as hyperbilirubinemia and it is a yellow discoloration of human tissues caused by the buildup of excess bilirubin. (1) Bilirubin deposition occurs only when bilirubin levels are high, indicating increased synthesis or decreased excretion. Bilirubin serum levels are typically less than 1 milligrams per deciliter (mg/dL). However, the clinical manifestation of jaundice with peripheral yellowing of the eye sclera, also known as scleral icterus, is most noticeable when serum bilirubin levels exceed 3 mg/dl. With an increase in serum bilirubin levels, the skin may gradually discolor, ranging from lemon yellow to apple green, especially if the process is prolonged; the green color is due to biliverdin. This exercise looks at the examination and differential diagnosis of jaundice. (2)

Importance

1. Better Treatment: Helps create effective medicines for jaundice.

2. Improved Formulas: Finds the best way to mix ingredients for better results.

3. Safety First: Ensures medicines are safe for patients.

4. Better Patient Care: Leads to more effective treatment and healthier patients.

Objectives of paper

1. Improve Emulsion Formulation: Identify optimal analytical techniques to enhance emulsion stability and efficacy.

2. Standardize Testing Methods: Establish standardized protocols for testing emulsions used in jaundice treatment.

3. Enhance Patient Safety: Evaluate analytical techniques to ensure the quality and safety of emulsion-based medications.

• Anatomy and Physiology                                                                      

Physiology of bilirubin metabolism

There are two forms of bilirubin: direct (water-soluble) and indirect (water-insoluble). Identifying the onset of each type is crucial in determining the type of jaundice. It is crucial to understand the natural process of bilirubin synthesis and clearance from the gastrointestinal tract. The spleen breaks down fragile and senescent red blood cells (RBC). When the RBC membrane ruptures, haemoglobin is liberated. Macrophages in the reticuloendothelial system phagocytose this. Haemoglobin is further separated into heme and globin. Heme is broken down into free iron, which interacts with ferritin and four pyrrole nuclei to produce bile pigments. (3)  Biliverdin is produced and subsequently transformed into bilirubin. Bilirubin is liberated into the plasma. It binds to albumin in plasma and travels to the liver. It is absorbed across the hepatic cell membrane. During this process, it is liberated from albumin but remains connected to Y and Z proteins within hepatocytes. Bilirubin quickly separates from the proteins and becomes conjugated. The final products are bilirubin glucuronide (80%), bilirubin sulphate10% Class I other, 10% other an active transport process and then excretes bilirubin glucuronide to the bile canaliculi. Some conjugated bilirubin spills into the plasma. It inters into the intestine through extrahepatic biliary pas- sages. Bacteria acts on conjugated bilirubin and forms urobilinogen. (3) Conversion of urobilinogen to its oxidized forms to stercobilinogen which is secreted into the feces) as stercobilin. The yellowish colour of normal stools has been attributed to the presence of stercobilin. It absorbs a section of urobilinogen and consequently, it is passed to the kidneys from where it's excreted as a colored pigment termed urobilin into urine which gives to normal urine a yellowish color. (3)

Anatomy                                                                                                                                 The biliary tree's intricacy can be reduced to much simpler portions. The intrahepatic ducts merge to form the right and left hepatic ducts, which exit the liver and connect to form the common hepatic duct. The cystic duct separates from the main hepatic duct and drains into the gallbladder. After the cystic duct exits, the common hepatic duct becomes the common bile duct (CBD) and leads to the duodenum (4). The CBD connects to the pancreatic duct and drains into the duodenum's second part. Obstruction, inflammation, or infection of the bile ducts alters their basal structure and size. The CBD and pancreatic duct have normal calibers of less than 8mm and 4mm, respectively. The gallbladder wall is normally <4mm thick. Rokitansky-Aschoff sinuses are epithelial invaginations on the gallbladder wall caused by increased pressure. (4)
The Luschka ducts are biliary ducts located in the gallbladder fossa, connecting the liver and gallbladder. These can lead to bile leaks following cholecystectomy. (4)

Normal bilirubin Levels:

Total bilirubin:

-Newborns (0 to1 week): 1.4-12 mg/dL
- Adults: 0.1 to 1.2 mg/dL

Direct (Conjugated): -

For newborns, bilirubin levels range from 0.1 to 0.4mg/dL.
- Adults: 0.1 to 0.3 mg/dL

Indirect (unconjugated) Bilirubin:

- Newborns: 1 to 10 mg/dL

- Adults: 0.2–0.8 mg/d

• Types of Jaundice                                                                                                        

Pre-hepatic:

Pre-hepatic jaundice is a kind of jaundice induced by haemolysis, known as haemolytic jaundice. Red blood cell plasma membrane defects are a significant cause of increased haemolysis. The cell membrane is unable to survive shear stress, leading to haemolysis and elevated serum bilirubin levels. Elevated urobilinogen and normal or higher urine bilirubin may indicate hepatocellular or red cell breakdown, such as haemolytic jaundice. Obtain a complete blood count, including reticulocyte count and peripheral blood smear, to identify haemolysis.

Causes: Pre-hepatic jaundice is typically caused by haemolytic anemia, which leads to excessive heme breakdown.

Symptoms: Patients with haemolytic jaundice may exhibit anemia, yellowing of the sclera, dark yellow-brown urine, yellowish skin, and elevated bilirubin levels. (5)

Hepatic:

Hepatic jaundice occurs due to a fault in the liver's hepatocytes. The liver absorbs bilirubin from plasma proteins, primarily albumin, and excretes it through the biliary system. Hepatic jaundice can be caused by liver disease that disrupts the capture, conjugation, and excretion processes. UDP-Glucosyltransferase is the main enzyme responsible for conjugation. Neonatal Physiological Jaundice can result from under-activity of this immature system at birth. This enzyme may be faulty because of genetic mutation in the UTG1A gene on chromosome 2. The gene for UDP-Glucosyltransferase causes hepatic jaundice when the conjugating enzyme is not functioning properly. Hepatic jaundice can result from a bilirubin excretory defect. The excretory systems are hepatocytic bile acid-independent, hepatocytic bile acid- dependent, and bile ductular secretion. Defects in excretory processes can cause bilirubin to accumulate in the blood, leading to hepatic jaundice.

Causes: Hepatic jaundice occurs when the liver fails to collect, conjugate, and excrete bilirubin properly.

Symptoms: Hepatic jaundice can cause abdominal pain, fever, vomiting, and nausea, as well as gastrointestinal bleeding, diarrhea, anemia, edema, weight loss, and weakness. If left untreated, it can lead to mental disturbances like kernicterus, coma, or death. (5)

Post-hepatic:

In post-hepatic jaundice or obstructive jaundice, there is an impediment to the flow of bile due to a partial or complete obstruction of the extrahepatic biliary passage between the liver and duodenum. Obstruction can occur within the biliary ducts themselves or more distal within the pancreas. This is predominantly a conjugated hyperbilirubinemia.

Causes: Extra-hepatic biliary blockage is the most common cause of post-hepatic jaundice.

Symptoms: Obstructive jaundice can present with dark urine, pale stools, and pruritus. Obstructive jaundice might be indicated by symptoms such as fever, biliary colic, weight loss, abdominal discomfort, or abdominal mass. Obstructive jaundice can cause problems such as cholangitis, pancreatitis, kidney and liver failure. (5)

• Diagnosis: (Laboratory tests)

Liver Function Test: Liver function tests allow your doctor to monitor your liver's health and detect liver disease. These blood tests assess the quantities of certain proteins and enzymes in your bloodstream. Proteins are big molecules that are essential to your general health. Enzymes are biological proteins that facilitate crucial chemical processes in your body. (8)

Bilirubin levels: Liver biochemistry tests for individuals with jaundice or cholestasis include total and direct bilirubin levels, aspartate transferase, Alanine aminotransferase, Gamma-glutamyl transferase, and alkaline phosphatase (ALP) values. A low blood GGT level that is disproportionate to the degree of cholestasis may indicate hereditary cholestasis, such as inborn errors in bile acid production. Biochemical analysis can help diagnose some metabolic problems. Mass spectrometry is used to analyze diseases like IEBAM (inborn error of bile acid metabolism) and metabolic abnormalities like NICCD (Neonatal intrahepatic cholestasis caused by citrin deficiency which blocks the flow of bile).

Liver Enzyme Tests

Alanine transaminase (ALT) is an enzyme found mostly in the liver. The ALT test examines the amount of ALT in your blood. Consistently elevated ALT levels in your blood might indicate liver disease.  Aspartate Transaminase (AST)is an enzyme found in abundance in the liver and other regions of the body. The AST test examines the amount of AST in your blood. High levels of AST might indicate liver disease.  Alkaline Phosphatase (ALP) is an enzyme found in abundance in your liver, bile ducts, and other organs. The ALP test detects the amount of ALP in your blood. High ALP values may indicate liver or bile duct disease. (8)

Liver Protein Tests: Total Protein refers to the quantity of protein in your blood. The two most common proteins present in blood are globulins and albumin. Globulin is a protein produced in your liver that aids the immune system in fighting infections. Low globulin levels may indicate liver injury or other problems. Albumin is another protein produced in the liver. An albumin test assesses how well your liver produces the proteins your body requires. Low albumin levels might be indicative of liver injury.  Prothrombin is a liver-produced protein that aids in blood coagulation. A prothrombin time test evaluates how long it takes for your blood to clot. A high prothrombin time might indicate liver injury. (8)

Genetic Test: Over the last two decades, technological advancements in genetic analysis have led to significant evolution in genetic tests. Conventional genetic diagnosis involves sequencing specific genes depending on the patient's phenotype. In 2007, a resequencing chip was designed to discover 5 genes associated with hereditary cholestasis. Denaturing high-performance liquid chromatography and high-resolution melting analysis can discover single-gene variations in a large number of patients. NGS panels for liver illnesses have included a restricted number of genes, including PFIC. Clinical trials using expanded panel-based NGS with over 50 genes have yielded encouraging results. Whole exome sequencing was used to find new disease-causing genes.

Table 1: Differential diagnosis of jaundice

Ultrasonography, computed tomography, and magnetic resonance cholangiopancreatography are three noninvasive imaging techniques for people with jaundice. Ultrasonography or computed tomography is typically the first-line option for evaluating blockage, cirrhosis, and vascular patency, with ultrasonography being the least intrusive and most cost-effective method. Magnetic resonance cholangiopancreatography or endoscopic retrograde cholangio- pancreatography can be used to see the intra- and extrahepatic biliary tree further, with the latter allowing for treatment options such as biliary stent implantation to ease blockage. Endoscopic ultrasonography, in conjunction with endoscopic retrograde cholangiopancreato- graphy, can evaluate common bile duct blockages and distinguish between a mass and a stone.

Fig 1. Ct Scan                           

Fig 2. Ultrasound                     

Fig 3. Mri  

• Quality Assurance and Quality Control

Quality assurance (QA)

Quality assurance (QA) in emulsions include developing procedures, creating standards, auditing and documenting processes, preventing mistakes, and taking corrective measures to assure consistency, dependability, and regulatory compliance.

Quality Control (QC)

To assure safety, emulsion quality control includes testing and validating the finished product's physical, chemical, and microbiological qualities to defined requirements, such as viscosity, pH, particle size, and stability evaluations.

• Regulatory Guidelines

Regulatory guidelines for emulsions are crucial to ensure the safety, quality, and efficacy of emulsion-based products, including pharmaceuticals and food items. These guidelines provide a framework for manufacturers to follow, guaranteeing compliance with strict standards and protecting public health.

US FDA:

1.Title 21 of the Code of Federal Regulations (CFR): This comprehensive rule sets out detailed criteria for drug products, including emulsions.
2.FDA guidance documents: Consult FDA guideline publications on specific issues, including as stability testing, manufacturing methods, and quality assurance. (10)

ICH Guidelines:

ICH Q1A(R2): Offers recommendations on the stability assessment of novel pharmacological substances and products.
ICH Q3A(R2): Outlines impurity testing recommendations that are useful for managing contaminants in emulsions.
ICH Q8(R2): Guidelines for pharmaceutical development that can be used to emulsion development. (11)

WHO Guidelines:

The WHO Technical Report Series: It produces a variety of technical publications on pharmaceutical production methods, including emulsion recommendations. WHO Good Manufacturing Practice (GMP): WHO GMP standards provide specific requirements for the production of pharmaceutical goods, including emulsions. (12)

• Good Laboratory Practices
• Laboratory Organization and Personnel
  1. responsibility: Establish the roles and duties of those involved in emulsion preparation and testing.
  2. Training: Ensure that people are taught in GLP principles, emulsion preparation, and testing protocols.
  3. qualifications: Check personnel credentials and experience. (13)
• Equipment and Instruments
  1. Calibration: Regularly calibrate equipment and instruments (such as pH meters and spectrophotometers).
  2. Maintenance: Follow the manufacturer's recommendations for servicing equipment and devices.
  3. Validation: Prepare equipment and instruments for specified testing.
• Characterization of both test and reference items
  1. Test Item: Determine the emulsion's composition, pH, and viscosity.
  2. Reference Item: Describe the reference item (such as standard emulsion). (14)
• IV: Standard Operating Procedures (SOPs)
  1. Preparation: Create SOPs for emulsion preparation, testing, and storage.
  2. Testing: Create standard operating procedures (SOPs) for analytical testing such as particle size and zeta potential.
  3. Data Management: Create SOPs for data management and reporting.
• Emulsion Preparation
  1. Formulation: Create emulsions using a proven formulation technique.
  2. Labelling: Emulsions should be labelled with their identification, date, and storage instructions.
  3. Storage: Keep emulsions under regulated conditions (such as temperature and humidity). (16)
• Analytical Testing.
  1. Particle Size: Determine particle size using proven methods (for example, dynamic light scattering).
  2. Zeta Potential: Determine zeta potential using approved methods (for example, electrophoretic light scattering).
  3. Viscosity: Determine viscosity using established methods (such as rotational viscometer).
  4. pH: Determine pH using proven methods (such as a pH meter).
  5. Stability: Assess stability under a variety of situations (such as temperature and humidity). (15)
• Different Analytical Techniques For Emulsion

Particle Size Analysis (PSA):

Particle size significantly influences emulsions' stability, texture, and bioavailability. Smaller particles minimize creaming and sedimentation, ensuring consistent texture. They also improve bioavailability of components, allowing the body to absorb essential nutrients more easily. Additionally, smaller particles increase surface area, facilitating chemical reactions and incorporating components like flavorings. Controlling particle size allows formulators to customize emulsions' stability, bioavailability, and reactivity to achieve desired product characteristics and performance.

Standard values:
For Pharmaceutical Emulsions:

- Average particle size is 1-5 μm (micrometers).

- Particle size range is 0.1-10 μm.

-  Polydispersity Index (PDI): <0.3 indicates

narrow distribution.

Procedure:

1. Sample Preparation:

- Dilute the emulsion with a suitable solvent (e.g., water, buffer) to a concentration of 0.1-1% (v/v).

- Use a sonicator or vortex mixer to ensure uniform dispersion.

2. Instrument Setup:

- Calibrate the instrument with a standard (e.g., polystyrene beads).

- Set measurement parameters (e.g., wavelength, scattering angle).

3. Measurement:

- Measure the particle size distribution using the chosen technique.

- Record data in triplicate.

4. Data Analysis:

- Calculate mean particle size (e.g., D50, D90).

- Calculate PDI and other relevant parameters (e.g., span, CV).

Instrument:

Dynamic Light Scattering (DLS) is a technique used to determine particle size and Polydispersity Index (PDI) in samples. It uses scattered light intensity to correlate with particle size, allowing researchers to characterize samples based on observed patterns. This method contributes significantly to understanding the physical properties and behavior of particles, making it a crucial tool in particle size analysis and nanomaterials and colloidal systems studies.

Example results:

Table 2

- Mean particle size: 2.5 μm (within the desired range).

- PDI: 0.2 (narrow distribution).

- Particle size range: 1-5 μm (suitable for emulsion stability). (17.18,19,20)

Viscosity Measurement

Viscosity plays a crucial role in determining the stability of an emulsion, influencing its overall texture and significantly impacting the bioavailability of its components.

Standard Values:

For pharmaceutical emulsions:

- Viscosity: 100-1000 mPa·s (millipascal-seconds) - Viscosity Index: 1-3

Procedure:
1. Sample Preparation: Make sure the emulsion is homogenous and free of air bubbles.
2. Instrument Setup: Calibrate the instrument using a standard (e.g., water or glycerin).
3. Measurement: Use the approach you've chosen to determine the viscosity.

4.   Data Analysis: Record viscosity values and

compute the viscosity index.

Instrument:

A specialized rotational viscometer called the Brookfield DV-II+ is used in labs to measure the viscosity and shear rate of various materials. It functions by measuring torque in relation to rotating speed, offering important information about the rheological characteristics and flow behavior of liquids and semi-solids. For quality control, research, and process optimisation, this easy-to-use tool is crucial in sectors including pharmaceuticals, food & beverage, and cosmetics. It helps with material selection, production procedures, and product formulations, enhancing product quality and guaranteeing regulatory compliance.

Example results:

Table 3

- Viscosity: 500 mPa·s (within the desired range).

- Viscosity Index: 2 (indicating non-Newtonian behavior).

- Shear Rate: 10-100 s-1 (suitable for emulsion stability).

- Flow Time: 30-60 s (indicating moderate viscosity). (21,22,23,24)

HPLC (High-Performance Liquid Chromatography)

High-performance liquid chromatography (HPLC) is a technique used to analyze emulsion components, including bilirubin. It helps scientists identify individual compounds, providing insights into their abundance and distribution. HPLC's precision and sensitivity enable detailed studies on bilirubin's impact on emulsion properties and stability. This analysis contributes to advancements in industries like pharmaceuticals, food, and cosmetics, enhancing knowledge of emulsion science and analytical chemistry.

Standard Values:

For pharmaceutical emulsions:

- Bilirubin concentration: 1-10 mg/mL

- Purity: ≥ 95%

- Impurities: ≤ 5%

Procedure:

1. Sample Preparation: Dilute the emulsion with a suitable solvent (e.g., methanol, acetonitrile).

2. Column Selection: Choose a suitable column (e.g., C18, C8).

3. Mobile Phase: Select a suitable mobile phase (e.g., water, acetonitrile, buffer).

4. Instrument Setup: Calibrate the instrument with a standard (e.g., bilirubin).

5. Measurement: Measure the chromatogram using the chosen detection method (e.g., UV, fluorescence).

6. Data Analysis: Calculate peak area, retention time, and concentration.

Instrument:

The Agilent 1260 Infinity II HPLC system, equipped with a UV-Vis detector and C18 column, is a reliable tool for precise compound separation and quantification, making it suitable for high-resolution analysis in pharmaceutical, environmental, and other fields.

Example results:

Table 4

- Bilirubin concentration: 5.0 mg/mL (below acceptable limits).
- Purity: 98.5% (meets requirements).

- Impurities: 1.5% (as required).

- Retention time: 10.2 minutes (compatible with standard).
- Peak area is 1000 mAU·s, showing appropriate sensitivity. ^{(25,26,27,28)}

UV-Vis Spectroscopy:

UV-Vis Spectroscopy is a crucial analytical technique for analyzing emulsion components like bilirubin, providing insights into chemical properties and concentrations. It aids in detecting liver conditions and disorders, making it essential in clinical chemistry and biomedical research.

Standard Values:

For pharmaceutical emulsions:

- Bilirubin absorption: 450 nm (maximum absorption wavelength)

- Absorbance: 0.5-2.0 AU (absorbance units)

- Specific absorbance: 10-50 AU/cm

Procedure:
1. Sample Preparation: Dilute the emulsion using a suitable solvent (such as water or buffer).
2. Calibrate the device using a standard (e.g., water or bilirubin).
3. Measurement: Take the absorption spectra across the selected wavelength range (e.g., 200-700 nm).
4. Data Analysis: Determine the absorbance, specific absorbance, and concentration.

Instruments:

The Agilent Cary 60 UV-Vis spectrophotometer, equipped with a quartz cuvette and Cary WinUV software, is a reliable instrument for analytical chemistry, providing accurate measurements and reliable results for complex experiments.

Example Results:

Table 5