A Comprehensive Review of Analytical Techniques for The Quantification of Salmeterol Xinafoate and Fluticasone Propionate in Pharmaceutical Preparations

Analytical chemistry, a subfield of chemistry, focuses on identifying components qualitatively and quantitatively and determining the amounts of substances, samples, or concoctions. Qualitative and quantitative analysis are the two types of analysis that are carried out: qualitative analysis focuses on identifying the components or analyte within concoctions or samples, whereas quantitative analysis is concerned with measuring the amounts of those components or analyte. [1]

Analytical Method

The term “analytical method” refers to the examining a sample to determine its qualitative, quantitative, or structural properties for one or multiple components analyte using a specific methodology and detailed, stepwise instructions. [2]. Analytical procedures: instrumental and classical approaches. (fig. 1). The classical approach involves a signal that correlates to the total amount of the analyte. A method is termed instrumental when the signal correlates with the concentration of the analyte.  [3].

Fig. 1. Classification of Analytical Methods

Instrumental methods can be broadly classified into four categories: spectroscopic, electrochemical, chromatographic, and other approaches. X-ray spectroscopy, NMR, Raman spectroscopy, IR spectroscopy, AAS and FES, and ultraviolet-visible spectroscopy are examples of spectroscopic techniques. Chromatographic techniques include GC, HPLC, TLC, column chromatography, paper chromatography and modern methods such as LC-MS, GC-MS, LC-MS-MS, GC-MS-MS, LC-NMR, and GC-NMR.[4]

Ultra-Visible Spectroscopy

Electromagnetic radiation and matter interact when matter absorbs and emits radiation (energy). Two kinds of spectroscopy exist: AAS and FES. Absorption spectroscopy refers to the study of electromagnetic radiation absorbed by a material, represented as spectra (Ultra-Visible, IR, NMR, microwave, and radiowave spectroscopy).[5] In ULTRA-VISIBLE-Visible spectroscopy, a sample or chemical compound absorbs ultraviolet or visible light, resulting in the emergence of various spectra. When molecules absorb ULTRA-VISIBLE light, electrons are excited and shift from lower to higher electronic energy levels. The subsequent ULTRA-VISIBLE emission spectrum is generated by the reverse transition. [5]

HPLC

Chromatography is a technique that separates mixtures of chemicals by employing both physical and chemical principles. Through the use of two distinct phases, the chromatographic technique is able to disassemble a mixture of many chemicals into two phases:  SP and  MP.[6] HPLC stands for high-performance or high-pressure liquid chromatography. HPLC can identify, isolate, and measure chemicals in any dissolved sample. [7] The core concept of liquid chromatography is adsorption. The MP in this chromatographic technique is a liquid. The sample takes on the characteristics of a liquid. In a column, An injection of a sample into a porous substance (SP) and a liquid phase (MP). A high pressure MP supplied by a pump is utilize in an example to pass over the column. The components of the sample move toward the SP in accordance with their affinity. Slower motion is exhibited by the component with a greater affinity for the SP. The SP has less attraction for the component that moves more quickly. The parts are isolated from one another.[8]

Analytic Methodology Development

The process of analytic methodology development involves choosing a precise assay methodology for ascertaining the composition of the formulation. Analytic Methodology Development is the procedure that demonstrates that an analytical method can be utilize in a lab. Analytical procedures that must be used in GMP and GLP environments should be designed in accordance with the protocols and acceptance criteria of the ICH recommendations. Q 2 (R1): [9]

To develop the method, the following criteria must be satisfied:

1. Analysts with qualifications

2. Instruments those are both qualified and calibrated

3. Methods that are documented

4. Reference standards that are reliable

5. Integrity and selection of samples

6. Control of change

The development of analytical method is beneficial for:

1. New processes and reactions

2. Development of new molecules

3. Macro analysis

 4. Micro analysis

5. Impurity profiling

6. Stress degradation studies

7. Herbal remedies [9]

Method Validation

In the United States, the idea of method validation was developed in 1978. Over time, the concept of method validation has broadened to include a variety of activities, such as computerized systems for clinical trials, process control, or labelling, as well as analytical procedures used to control the quality of medicinal components and products. Method validation is considered a vital and indispensable part of cGMP. The term "Method Validation" refers to the assessment of efficacy or validity. A team effort including individuals from various plant branches is method validation. Method validation is the process of "establishing documented evidence" that provides a high level of assurance that the product (equipment) will meet the requirements of the intended analytical applications.[10]

Significance of Method Validation

• Quality assurance
• Minimal failure rate for batches
• Decrease in rejections
• Efficiency and productivity enhancements
• Output escalation
• Reduction in testing of processes as well as end products

Parameters of  Method Validation

1. Accuracy
2. precision
3. Linearity
4. Limit of detection
5. Limit of quantitation
6. Specificity
7. Range
8. Robustness [10]

SX is chemically known as,2-(Hydroxymethyl)-4-[1-hydroxy-2-[6-(4-phenylbutoxy) hexylamino] ethyl] phenol; 1-hydroxy-2-naphthoic acid. SX is used to treat chronic obstructive pulmonary disease and asthma [11,12]. Formulated as its 1-hydroxy-2-napthoate (xinafoate) salt, it is a long-acting and highly selective β2 agonist that is used to treat chronic obstructive pulmonary disease and asthma.. SX is inhaled to address asthma flare-ups and, similar to other beta-2-agonists, induces bronchodilation through the relaxation of the airway's smooth muscle. The long-lasting effect takes place as the molecules first permeate the lung cells' plasma membrane, after which they are progressively released back into the extracellular space, enabling them to engage with the beta-2 adrenoceptors, while the lengthy carbon chain serves as a membrane anchor.  [13]  FP is a chemical compound with the name [(6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17(fluoromethylsulfanylcarbonyl)-11-hydroxy-10,13It is utilized for treating asthma, allergic rhinitis, and atopic dermatitis. [14,15] It is a trifluorinated corticosteroid with high potency based on the androstane structure, exhibiting neutral characteristics. Due to its anti-inflammatory properties, it is useful for treating asthma and allergic rhinitis.  Additionally, it is employed in treating eosinophilic esophagitis. FP imitates the hormone that occurs naturally and is generated by the adrenal glands, known as cortisol or hydrocortisone.  [4]

Recent updated studies on Analytical Methods

• Research by Mohammad Jamal A. Shammout et.al aimed to obtain a number of benefits, including low detection limits, short retention durations, and a straightforward, non-buffered MP. During solution preparation, this method utilizes a Ultra-Visible -1800 Ultra-Visible spectrophotometer to scan the Ultra-Visible spectrum and calculate the λmax. An I-Series LC-2030 High-Performance Liquid Chromatography (HPLC) system from SHIMADZU with Ultra-Visible detector was utilized for the chromatographic analysis. SX was detected at 252 nm, whereas FP was detected at 236 nm. For HPLC, an isocratic 50:20:30 (v/v) ratio of METH, ACN, and D/W served as the MP; for Ultra-Visible analysis, METH was utilized. A steady 1 mL/min FR was maintained. The HYPERSIL column had RT s of 10.6 minutes for FP and 1.8 minutes for SX, whereas the C18 SUPELCO column had RT s of 6.3 minutes for FP and 1.9 minutes for SX. The SUPELCO column was shown to require less RT than the HYPERSIL column, most likely as a result of the SP's more even packing in the former. The stability test was carried out in a number of scenarios. Under acidic (1M HCl) conditions, both FP and SX showed poor stability, with recoveries of less than 57%. In alkaline conditions (1M NaOH), FP was unstable at all concentrations with recoveries below 41%, whereas SX was stable only at higher concentrations (26.67 and 40 µg/mL) with recoveries of 100% and 113%, respectively." With recoveries not going above 70%, both medications were unstable in oxidative environments. [16]
• Serkan Acar et.al study utilizes a high-performance liquid chromatography (HPLC) approach with a MP made up of ACN and PDP buffer (pH 3.0). The system was run at a 1.5 mL/min FR while keeping the tray and column temperature at 25°C and 40°C, respectively. Ultra-Visible detection was done between 210 nm wavelength. A 40 µL injection volume was utilized, along with a stainless steel column (15 cm × 4.6 mm, 5 µm) filled with octadecylsilyl silica gel (Hypersil BDS). In order to guarantee efficiency and quick throughput, the entire analysis time was optimized to 10 minutes. ICH Q2 (R1) recommendations were followed in the method's rigorous Method Validation, which included a thorough assessment of numerous important characteristics, such as specificity, linearity, limit of quantitation (LOQ), accuracy, precision, robustness, and the stability of the solution and MP. The approach satisfies the strict requirements outlined by ICH guidelines and is accurate, exact, robust, specific, and selective, according to the Method Validation process results. The Method Validation results demonstrate the devised method's robustness and dependability, proving its appropriateness for medicinal analysis. This work highlights HPLC's potential for usage in high-standards analytical applications and makes a substantial contribution to the methodology's advancement.[17]
• For ensuring the reliability for quantifying both drugs even in the presence of degradation products K.R. Wagh et.al devised a technique for the simultaneous estimation of SX and FP in bulk and pharmaceutical dose forms using reverse-phase high-performance liquid chromatography (RP-HPLC). An octadecyl C18 column (5 μm, 25 cm × 4.6 mm, i.d.) was used to accomplish the chromatographic separation, and the MP was made up of METH and D/W in a 70:30 ratio at pH 3 that was adjusted using orthophosphoric acid (OPA). The ULTRA-VISIBLE detection was carried out at 232 nm with the FR set to 0.8 mL/min. Twenty microliters was the injection volume. It was discovered that the RTs for SX and FP were 3.59 and 6.73 minutes, respectively. The ICH Q2 (R1) guideline was used to verify this approach as stability-indicating.[18]
• Shubhangee Gaikwad et.al research explores three different Ultra-Visible spectrophotometric methods for the synchronous estimation of SX and FP in bulk and capsule dosage forms:

The method I: Simultaneous equation method:

Both drugs' wavelengths were chosen for quantification based on their overlain spectra: 236 nm for FP and 216.5 nm for SX.

Method II: Absorbance ratio method (Q-Analysis)

The absorbance ratio approach makes use of the ratio of absorbances at two specific wavelengths, one of which is the λ max of one of the two components and the other is an isoabsorptive point. It is clear from the two medications' overlay spectra that FP and SX exhibit an isoabsorptive point at 262.5 nm.

Method III: Area under the curve

For both medications, the area under the curve was calculated at the chosen wavelengths between 214 and 218 nm (SX) and 234 and 238 nm (FP). The wavelengths selected for the analysis were 216.5 nm for SX (SX) and 236 nm for FP (FP). These methods provide accurate and reliable results for the synchronous measurement of the two drugs within their medicinal formulations.[19]

• Arzu Çay?r et.al successfully designed an analysis method using an Agilent HPLC system with an autosampler, quaternary gradient pump, and online degasser. A PDA detector was utilize to detect the signals. The SP, a Hypersil BDS C8 column (15 cm x 4.6 mm, 5 µm, Thermo Fisher Scientific, Runcon, UK) was kept at a constant temperature of 30ºC in a thermostatically controlled oven. The MP contained two solvents: solvent A, a 0.1 M ammonium phosphate buffer (11.5 g of ammomium dihydrogen phosphate diluted in 100 mL of D/W, pH adjusted to 2.9 OPA), and solvent B, ACN. The FR was set at 2 mL/min, with an injection volume of 20 µL and detection at a wavelength of 228 nm. With the help of this technique, related chemicals in the dry powder inhaler formulation of FP and SX were successfully identified.[20]

• M. Shahanaz et.al developed a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the synchronous quantification of FP and SX in both bulk and medicinal dosage forms. The analysis was conducted using an X-Terra C18 column (4.6 × 250 mm, 5 µm particle size), with a MP composed of METH, ACN, and D/W in a 50:35:15 (v/v) ratio. The FR was maintained at 1.0 mL/min, and detection was performed with a (PDA) detector at 280 nm. The RT s for FP and SX were 1.6 minutes and 3.2 ± 0.02 minutes, respectively. The method was validated for accuracy, precision, and reliability, confirming its suitability for routine analysis of these drugs in medicinal formulation.[21]
• For discriminating the gradient and isocratic elution E. P?czkowska et.al conducted  research which utilized a reverse-phase high-performance liquid chromatography (HPLC) method to analyse SX and FP in dry powder inhaler products. The chromatography was performed using a C8 Luna column (100 × 4.6 mm, 5 µm particle size) in gradient mode. The MP consisted of two components: a buffer solution (0.6% trifluoroacetic acid in D/W-tetrahydrofuran, 8:2 v/v) and a concoction of ACN and METH (1:1 v/v) in a 60:40 ratio. The FR was set at 1.5 mL/min, and the detection was carried out using a Ultra-Visible detector at wavelengths of 239 nm and 250 nm. The ChemStation Plus Method Validation Pack software (Agilent Technologies, Santa Clara, USA) was utilize to validate the procedure. In comparison to isocratic elution, the gradient elution mode yielded smaller peaks and faster analysis. This technique is appropriate for quality control of dry powder inhalers since it also ensured the effective and quantitative recovery of both active components through the use of DMSO (dimethyl sulfoxide) as a solvent in concoction with METH and D/W.[22]
• In order to use reverse-phase liquid chromatography (RP-LC) in conjunction with ultraviolet (Ultra-Visible) detection and a forced degradation study, Bediha Akmesea et.al carried out a work. Using a Diode Array Detector (DAD) system (SPDM 20A), the wavelengths for the detection of SX and FP were set at 210 and 238 nm, respectively. ACN (ACN) and D/W were combined at 55%, 60%, and 65% (v/v) with 25 ml phosphoric acid to create an isocratic MP, which was utilize in the chromatographic analysis. The column was an XTerra RP18 (250 × 4.60 mm ID, 5 µm). Using sodium hydroxide, the MP's pH was changed from 3 to 12. A 20 µL injection volume and a 1.0 mL/min FR were established. The stability of FP and SX was assessed in the forced degradation study under a range of stressors, such as hydrolytic, oxidative, thermal, and photolytic stress. The findings revealed notable deterioration in the following conditions:

• HCl (1 M): No degradation for FP; 91.1% degradation for SX.
• NaOH (1 M): 99.8% degradation for FP; no degradation for SX.
• H₂O₂ (3%): 73.0% degradation for FP; 47.0% degradation for SX.
• H₂O₂ (30%): 94.4% degradation for FP; 75.1% degradation for SX.
• ULTRA-VISIBLE exposure (24 hours): No degradation for FP; 47.5% degradation for SX.
• Heating at 75°C: No degradation for either compound.
• Heating at 100°C: No degradation for either compound.

This approach shows advantages over previously published methods in terms of recovery, reproducibility, sensitivity, and the use of less expensive reagents, making it a more effective and economical method for analysis.[23]

• Novel spectrophotometric method was introduced by Ahmed Samir et.al for the synchronous determination of SX and FP. The method employs Ultra visible spectrophotometry, utilizing both the first derivative spectra and zero-order absorption spectra for analysis. The wavelengths selected for detection were 225 nm for SX (SX) and 256.5 nm for FP (FP). This method offers a simple, effective approach for analyzing these compounds in bulk powder and inhaler formulations. [24]
• Four different approaches are presented by Ahmed Samir et.al research for concurrently identifying FP and SX in bulk powder and inhaler goods. The first technique makes use of a reversed-phase column and High-Performance Liquid Chromatography (HPLC) with a MP consisting of ACN and METH in an 80:20 (v/v) ratio. Both medicines were separated using a FR of 0.5 mL/min and Ultra-Visible detection at 220 nm. The RT  for SX is 2.610 and for FP is 3.390. In the second technique, zero-crossing observations are made at 269.5 nm for FP and 352 nm for SX using first derivative ULTRA-VISIBLE spectrophotometry. The third method applies the first derivative of the ratio spectra, where the amplitudes are measured at 334 and 337.5 nm for SX, and at 225 and 231.5 nm for FP. The fourth method uses the isobestic point at 237.5 nm. SX is determined by measuring its ULTRA-VISIBLE absorbance at 343 nm, which is unaffected by FP. The study also includes a comparative analysis of the HPLC and spectrophotometric techniques for resolving the two drugs in their concoctions. The authors note that while the spectrophotometric methods (D1, DD1, and isobestic point) provide high resolution, faster analysis, and lower costs, HPLC offers greater specificity. However, HPLC requires more expensive equipment and materials. In contrast, the spectrophotometric methods are simpler, more cost-effective, and suitable for routine analysis.[25]
• The study by M.S. Kondawar et.al offers a ULTRA-VISIBLE spectrophotometric technique for the synchronous measurement of FP and SX in medicinal dosage forms and bulk. A solvent concoction consisting of 95% ethanol and phosphate buffer (pH 7.4) in a 90:10 ratio was utilize to perform the analysis. FP was detected at 246 nm, while SX was detected at 214 nm. The synchronous quantification of these two substances in their formulations is made easy, dependable, and efficient by this technology.[26]
• To get the reliable and efficient results for determining both drugs in the inhaler formulation Lantider Kasoye et.al developed a new High-Performance Thin-Layer Chromatography (HPTLC) for the synchronous analysis of SX and FP. The MP for the separation process consisted of a concoction of n-hexane, ethyl acetate, and acetic acid in a ratio of 5:10:0.2. Detection was carried out using a TLC scanner at a wavelength of 250 nm, providing a densitometric analysis of the compound. The Rf value for SX is 0.4± 0.04 and for FP is 0.6± 0.03 [27]

Table 1: Recent updated research works on estimation of SX and FP in concoction using HPLC and ULTRA-VISIBLE methods