1. A method of detecting Sarecycline hydrochloride in a biological sample comprising: a) adding internal standard of minocycline to a sample comprising sarecycline hydrochloride; b) using a liquid-liquid extraction technique to extract and purify the sarecycline and minocycline from human plasma; with methyl tertiary butyl ether as the extracting solvent; c) reconstituting the extracts using the eluent - ammonium formate: acetonitrile and injecting the same in HPLC-MS/MS system; d) Infusing the eluted solutions of seracycline and minocycline in ESI source of Mass spectrometer; and e) Analysing of the mass spectrometric data to detect and quantify the concentration of seracycline hydrochloride in biological sample.

2. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein the peak height of the seracycline hydrochloride is quantified using a calibration curve, said calibration curve is obtained by plotting data points of known concentrations of seracycline versus a peak height ratio of minocycline (internal standard).

3. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein the retention time of seracycline and minocycline were found to be 1.5 ± 0.2 min, with overall runtime of 3.0 min.

4. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein mass spectrometric analysis using Electron Spray Ionization yielded high-abundance fragment ions at m/z 488.6 / 291.1 and 458.4 / 337.2.

5. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein a highly sensitive and accurate method validation procedure was developed for determining Seracycline in human plasma for clinical / bioavailability and Pharmacokinetic study.

[001] The present disclosure relates to methods for quantitatively determining the presence and/or amount of a Seracycline hydrochloride in a solution by forming a corticosteroid-acetate adduct and detecting the adduct using a LC/MS/MS system. These methods accurately detect trace amounts of Seracycline.
BACKGROUND
[002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[001] Bioanalytical methods are sophisticated methods used for the quantitative determination of drugs in biological fluids plays a significant role in the evaluation and interpretation of bioequivalence, pharmacokinetic and toxicokinetic studies. Knowledge on drug concentration levels in body fluids, such as whole blood, plasma, serum, and urine, allows the optimization of pharmacotherapy and provides a basis for studies of patient compliance, bioavailability, pharmacokinetics and the influences of co-medications. Selective and sensitive analytical method development become necessary during the quantitative and qualitative analysis of drugs and their metabolites that are supposed to display pharmacological activity, determination of multiple drugs in combating a disease, biotransformation investigation, drug monitoring for therapeutic benefits and for in-vitro experiments
[002] quick and accurate determination of drugs within human plasma is of paramount importance in many medical applications. Methods that accurately and quickly determine even trace amounts of a drug in the bloodstream are particularly useful. Moreover, these detection methods become of particular importance in the use of high potency drugs because unwanted side effects are produced when safe dosage levels are exceeded. Consequently, research has focused on developing drug detection and/or quantification methods that concurrently analyze samples such as plasma for a variety of compounds in differing amounts
[003] Seracycline is a narrow-spectrum tetracycline-derived antibiotic. It is specifically designed for the treatment of acne, and was approved by the FDA in October 2018 for the treatment of inflammatory lesions of non-nodular moderate to severe acne vulgaris in patients 9 years of age and older.
[004]
Seracyclin hydrochloride
[005] Determination of drugs and their metabolites is complicated in biological matrix compared to in formulations. Biological matrix (e.g. blood, plasma, serum and urine) samples contain mostly water and other components like dissolved proteins, glucose, clotting factors, mineral ions, hormones and acids. Drug absorption in body depends upon the properties of drugs and also some patient-related factors, therefore, it’s not possible all the time to avail high drug concentration in biological samples. Also, above mentioned components may interfere during the time of quantification of analyte of interest. There are a number of techniques used for the separation of drugs from biological matrices for analytical purposes. Methods generally used in the bioanalysis of drugs and their metabolites are radioimmunoassay (RIA), capillary electrophoresis (CE), gas chromatography (GC), GC-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC) with UV, fluorescence, refractive index and mass spectrometry detection (MS).
[006] HPLC is the technique for chemical and pharmaceutical analysis with the ability to separate, analyze, and/or purify the given sample [6].The separation of analytes isbased on differences in relative rates of migration through the column arising from different partition coefficient of the analytes in the stationary and the mobile phase. Reverse phase HPLC having hydrophobic stationary phase and polar mobile phase is generally used for the analysis of most of the compounds
[007] Liquid chromatography is a technique for separating components in a sample mixture. At any given time during separation, some molecules of a component are adsorbed to a stationary solid support, while other molecules are dissolved in a liquid solvent flowing past the solid support. The adsorbed molecules are said to be in a “stationary phase” while the dissolved molecules are said to be in a “mobile phase.” Separation is based upon the differences of the components' chemical and/or physical properties. Sample components can differ significantly in their solubility in a given solvent. Specifically, nonpolar components tend to dissolve more readily in organic solvents, while polar components tend to dissolve more readily in water. To accommodate samples with both polar and nonpolar component, reverse-phase gradient-elution liquid chromatography (GELC) provides for a gradual transition of organic solvent to water as the liquid solvent in an LC system.
[008] MS provides a fingerprint mass spectrum, which contains the molecular weight and structure-specific fragmentation of ion information. The major drawback of MS is its limitation in handling mixtures of compounds [10]. Tandem mass spectrometry (MS/MS) can lessen this problem to certain extent. Interfacing of HPLC with MS thus benefits mutually from the high-resolution separation capability of HPLC and highly sensitive and structure-specific detection capability of MS. MS comes very close to being a universal detector for LC[11].Sample preparation plays a important role in attaining high selectivity and sensitivity. It is mandatory to clean the biological sample as much as possible to get rid of matrix interferences free sample solution. A proficient extraction procedure is the need of hourfor developing and delivering quantitative and reproducible recovery of the given sample. Sometimes, concentrating the sample after extraction, derivatization at sample processing step or at chromatography phase and adduct ion formation can enhance the sensitivity of the method
[009] Thus, developments of selective and sensitive analytical methods for the quantitative evaluation of drugs are critical for the successful accomplishment of pre- clinical and clinical pharmacology studies These requirements can be achieved by HPLC, especially if combined with an advanced detection technique such as mass spectrometry (MS)
[0010] Now-a-days, analysis time of biological samples can be decreased sharply using a variety of analytical columns, but the choice of an appropriate sample preparation method is crucial for reliability and accuracy of the analysis as separation of analytes from other matrix components on column takes a short time. Prior to LC analysis, biological samples must be ‘cleaned up’. This involves isolation of the drug to be analyzed from its matrix with consistent recovery.Three sample preparation techniques are commonly used to extract analytes from biological matrices: protein precipitation, liquid-liquid extraction and solid phase extraction.
[0011] Liquid-liquid extraction (LLE) is still one of the most popular extraction techniques. This method is used to separate compounds based on their relative solubility in two different immiscible liquids, usually water and organic solvent. Sometimes it is required to back extract the compounds or multiple extractions to remove the interferences from the sample. It is cost effective method compared to solid phase extraction (SPE) but is tiresome and time consuming, often requires drying followed by reconstitution. LLE is a simple and proficient method for the separation and concentration of relatively hydrophobic compounds. For some polar compounds, it is not possible to get matrix free clean sample using this extraction procedure
[0012] Therefore, the present disclosure overcomes the above-mentioned problem associated with the traditionally available methods or systems, any of the above-mentioned inventions can be used with the presented disclosed technique with or without modification. All research publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies. Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
[0013] The Literature survey indicates that there are very few methods for the determination. There are several methods available with other drug combinations. Therefore, an attempt is made to develop method for preparation and stability of different salts of sulfathiazole using various acids and ammonium compounds.
[0014] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

OBJECTS OF THE INVENTION
[007] It is an object of the present disclosure is to a method of detecting a Seracycline HCl in a sample by adding an internal standard to a sample suspected of containing a seracycline.
[008] Another object of the present disclosure is removing, if necessary, interfering compounds from the sample; placing the sample on an HPLC column equilibrated with an ammonium acetate: acetonitrile as eluent
[009] Yet another object of the present disclosure to analyze the eluent of the HPLC column with a MS, wherein if contained in the sample, the SCL is detected by the MS.
[0010] Yet another further object of the invention is development of method validation for the aforementioned detection method.
SUMMARY OF THE INVENTION
[0011] The present disclosure relates to a method detecting Seracycline HCl in a sample by adding an internal standard to a sample suspected of containing a seracycline.
[0012] The present disclosure also n another embodiment of the invention, the internal standard is minocycline
[0013] According to the present disclosure; the interfering compounds are inorganic salts, organic materials, physiological materials, or combinations thereof. The interfering compounds are removed using liquid—liquid extraction, solid phase extraction, protein precipitation, or a combination thereof, preferably solid phase extraction.
[0014] The present invention involves a method used to separate compounds based on their relative solubility in two different immiscible liquids, usually water and organic solvent. Sometimes it is required to back extract the compounds or multiple extractions to remove the interferences from the sample. It is cost effective method compared to solid phase extraction (SPE) but is tiresome and time consuming, often requires drying followed by reconstitution. LLE is a simple and proficient method for the separation and concentration of relatively hydrophobic compounds. For some polar compounds, it is not possible to get matrix free clean sample using this extraction procedure.
[0015] As used herein, unless otherwise specified, the term “LC/MS/MS” or “LC/MS” means either a multiple analytical apparatus having a liquid chromatography and mass spectrometry or separate liquid chromatography and mass spectrometry apparatus used in sequential order, but not necessarily immediately thereafter.
[0016] One should appreciate that although the present disclosure has been explained with respect to a defined analytical method validation of developed stability indicating method, all of which also completely within the scope of the present disclosure. Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, explain the principles of the present disclosure.
[0018] FIG. 1 illustrates a Mass spectrum of Sarecycline (SC) Q1 and Q3 ions
[0019] FIG 2. illustrates a Mass spectrum of Minocycline (MN) Q1 and Q3 ions
[0020] FIG 3.: Blank plasma chromatogram of interference free Sarecycline HCl and Minocycline HCl
[0021] FIG 4: Calibration curve of Sarecycline HCl.
[0022] It should be noted that the figures are not drawn to scale, and the elements of similar structure and functions are generally represented by like reference numerals for illustrative purposes throughout the figures. It should be noted that the figures do not illustrate every aspect of the described embodiments and do not limit the scope of the present disclosure.
[0023] Other objects, advantages, and novel features of the invention will become apparent from the following detailed description of the present embodiment when taken in conjunction with the accompanying drawings.
DETAILED DESCRIPTION OF THE INVENTION
[0024] In the following description, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present invention. It will be apparent to one skilled in the art that embodiments of the present invention may be practiced without some of these specific details. As the detailed description is concerned various stages are included in embodiments of the present invention, which will be detailed below. The stages can be carried out along with statistical data and by machine-executable instructions, which can be used to direct a general-purpose or special-purpose processor to carry out the procedures. If the specification states a component or feature "may", "can", "could", or "might" be included or have a characteristic, that component or feature is not required to be included or have the characteristic.
[0025] Aspects of the present disclosure relate to a novel synergistic herbal formulation and a process for preparation of synergistic formulation using Indian medicinal herbs.
[0026] It is inferred that the foregoing description is only illustrative of the present invention, and it is not intended that invention be limited or restrictive thereto. Many other specific embodiments of the present invention will be apparent to one skilled in the art from the foregoing disclosure. All substitutions, alterations and modifications of the present invention which comes within the scope of the following claims are to which the present invention is readily susceptible without departing from the spirit of the invention. The scope of the invention should therefore be determined not with reference to appended claims but along with the full scope of equivalents to which such claims are entitled.
[0027] Thus, for example, it will be appreciated by those of ordinary skill in the art that the diagrams, schematics, illustrations, and the like represent conceptual views or processes illustrating systems and method embodying this invention. Those of ordinary skill in the art further understand that the exemplary processes, method, and/or pharmaceutical components described herein are for illustrative purposes and, thus, are not intended to be limited to any named.
[0028] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0029] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0030] In an embodiment of the present disclosure, the The development of a LC-MS/MS method required three separate methodologies to be developed
• Sample preparation
• Chromatography
• Mass spectrometry
[0028] In the given embodiment of the invention, Sarecycline.HCl and Minocycline.HCl was obtained from clear synth Labs (P) Ltd, Mumbai, India. Human plasma (K2EDTA), obtained from Navjeevan blood bank, Hyderabad. Formic acid, Ammonium formate, sodium carbonate, acetonitrile, methanol obtained from SD- Fine chemicals, Mumbai. MTBE (methyl-tertiary butyl ether) was obtained from Labscan,Mumbai. (Ultra pure water obtained from Milli-Q System.
[0028] API 4000 HPLC-ESI-MS/MS system (Applied Biosystems), 1200 Series HPLC system (Agilent Technologies, Waldbronn, Germany), data acquisition and processing were accomplished using Analyst® Software 1.4.1. Micro balance (ME5 model Sartorius), variable range micro pipette (Eppendorf), Autosampler vials (Agilent) ,variable size glass bottles, graduated measuring cylinders, volumetric flasks (Borosil), ultrasonic bath (Pharmatek Scientifics), Vortexer (Spinix), deep freezer (-30°C-Sanyo), nitrogen evaporator (Turbo Vap LV Caliper Life Sciences), refrigerator (LG). Pipette tips 10 µL-1000 µL, riavials, 12 x 7.5mm (Tarson) polypropylene tubes; combitips (Eppendorff) and variable size surgical gloves (Surgicare) were employed in the present investigation.
[0029] All the solvents used in this research were HPLC grade and regents were AR-grade. The volumes and concentrations were mentioned in the process are theoretical. The volumes and concentrations could be corrected on the basis of actual weights used.
Name of the solution Preparation
50% Methanol Mix 500 mL of methanol with 500 mL of water.
10mM Ammonium formate PH:4.5
Dissolve 1.26 g of ammonium formate into 2 L of water and adjust pH with formic acid

Autosampler wash Acetonitrile
Reconstitution solution 5 mM ammonium formate: acetonitrile (10:90v/v)
0.5NSodium carbonate Dissolve 26.5 g of anhydrous sodium carbonate into 1L of water.
Mobile phase 10mM Ammonium formate pH:4.5: Acetonitrile in the ratio of 50:50 and Filter through 0.45 µm filter
(Autosampler wash) 80% Acetonitrile Mix 800 mL of Acetonitrile with 200 mL of water.
Reconstitution solution Mix 500 mL of 10mM ammonium formate with 500 mL of acetonitrile.
Extraction Solvent MTBE (methyl-tertiary butyl ether)
Table. 01: Reagents and solvents preparation

[0031] In an aspect of the invention the Standard stock solutions of Sarecycline (100.0µg/mL) and Minocycline (100.0µg/mL) were prepared in methanol. The spiking solution for Minocycline (80.0 ng/mL) was prepared in 50% methanol from respective standard stock solution. Standard stock solutions and IS spiking solutions were stored in refrigerator conditions (2-8°C) until analysis. Standard stock solutions were added to drug-free human plasma to obtain Sarecycline concentration levels of 0.5, 1.0, 5.0, 15.0, 30.0, 45.0, 60.0, 90.0, 120.0 and 150.0 ng/mL for Analytical standards and 0.5, 1.5,75.0, and 105.0 ng/mL for Quality control standards and stored in a -30°C set point freezer until analysis. The Aqueous standards were prepared in reconstitution solution (10mM ammonium format: acetonitrile (50:50 v/v) for validation exercises until analysis.
[0032] In the present invention wide spectrum of organic solvents were tested from different physicochemical categories with different volume fractions as well as combinations. In terms of the analysis condition, various mobile phases, in different proportions, buffered and non-buffered at various pH were attempted to provide the best peak shape and less retention times.
[0033] In another aspect of the invention the Stability studies showed that there were no significant changes in physical and chemical properties of formulation F8 after 2 months. Tween 80 in 1:1 ratio (F8) was showing best release. F8 was compared with marketed and prepared conventional formulation and result shows better dissolution profile.
[0034] In another aspect of the invention, the MS optimization was performed by direct infusion of solutions of both Sarecycline and Minocycline into the ESI source of the mass spectrometer. The critical parameters in the ESI source include the needle (ESI) voltage, other parameters, such as the nebulizer and the desolvation gases were optimized to obtain a better spray shape, resulting in better ionization. A CAD product ion spectrum for Sarecycline and Minocycline yielded high-abundance fragment ions at m/z 488.6 / 291.1 and 458.4 / 337.2 (Figure 2 & 3) in multiple reaction monitoring (MRM) positive mode respectively. After the MRM channels were tuned, the mobile phase was changed from an aqueous phase to a more organic phase with acid dopant to obtain a fast and selective LC method.
[0035] In another aspect of invention, the most accurate extraction method for analyte was selected as Liquid-Liquid extraction. A good separation and elution were achieved using 10 mM ammonium formate (pH 4.5.): acetonitrile (50:50 v/v) as the mobile phase, at a flow-rate of 0.5 mL/min and injection volume of 10 µL. The notable advantages of the developed method are most sensitive and accurate methods over the developed methods based on literature.
[0036] In another embodiment, the sample preparation of seracycline and minocycline has been discussed. Liquid-liquid extraction was used to isolate Sarecycline and Minocycline from human plasma. 100 µL of Minocycline spiking solution (8.0 ng/mL) and 200 µL of plasma sample (respective concentration) were added respective ria vials and vortexed 30 seconds followed by 100 µl of 0.5N sodium carbonate solution was added and vortexed 10 minutes. Then, 2.5 mL of extraction solvent (Methyl Tertiary butyl ether) was added and vortexed approximately for 20 min. This was followed by, Centrifugation at 4000 rpm, 5 min at 20°C. Then samples were Flash freeze by using dry-ice/Acetone. The supernatant from each ria vial was transferred into another set of ria vials. These samples were evaporated at 40°C under nitrogen upto dryness. Finally, the dried residue samples were reconstituted with 500 µL of reconstitution solution (10mM Ammonium formate (pH 4.5): acetonitrile 50:50, v/v) and vortexed briefly. These samples were transferred into auto sampler vials and injected in to LC-MS/MS.
[0037] With the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions, or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
[0038] Thus, the scope of the present disclosure is defined by the appended claims and includes both combinations and sub-combinations of the various features described hereinabove as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.
EXAMPLE
BIOANALYTICAL METHOD VALIDATION
The objective of the work is to validate specific HPLC- MS method for the determination of Sarecycline in human plasma for clinical / bioavailability and Pharmacokinetic study.
Calibration Curve:
Quantifying the amount of corticosteroid wherein the peak height of the corticosteroid is quantified using a calibration curve. The calibration curved is obtained by plotting data points of known concentrations of SCl versus a peak height ratio of a known amount of corticosteroid/internal standard. Preferably, the calibration curve has from about 4 to about 9 data points. Calibration curve are shown in Figure 01 to Figure 02

System Suitability
Six replicate injections of aqueous mid QC along with internal standard were injected and the results were depicted in Table 68.

Table 02: System suitability of SC and MC
Injection number Peak area
Analyte (SC) Internal standard
(MC)
1 652340 422722
2 633232 412752
3 650322 432712
4 651341 432762
5 652340 432162
6 652345 432762
N 648653.33 427645.33
Mean 7597.85 8300.39
SD (±) 1.17 1.94
% CV (%RSD) 652340 422722

Acceptance criteria: The %CV (%RSD) for the analyte and internal standard should not be more than 5%.

[001] Limit of Quantification (LOQ) / Sensitivity
Six LLOQ standards were prepared in screened plasma lot along with IS (50.00 ng/mL) and signal to noise ratio (S/N) was calculated using analyst software Table 70.

Table 03: Limit of quantification of (Sarecycline HCl)
Plasma B. No LLOQ Peak area LLOQ S/N ratio
AP/3272/07/10
(Lot.No-2)
8174
70.6

8234
76.2

8078

8127
62.8

56.6

8133
58.0

8243
62.4

6
6
N
8164
64.43
Mean
8174
70.6

Acceptance criteria: The mean S/N ratio of LLOQ should be = 5.

[002] Recovery of (Sarecycline.HCl and Minocycline.HCl)
Standard
conc (ng/mL) Unextracted SC peak area Extracted SC
peak area Unextracted
MC
peak area Extracted
MC
peak area
Low QC
(11.5 ng/mL) 537130 527130 633388 653388
576252 556251 626057 606051
561091 561091 623125 613126
499819 409812 630394 630394
551165 511163 633630 663635
547313 527313 642957 672951
N 6.00 6.00 6.00 6.00
Mean 545091.40 515460.00 629318.80 639924.17
SD(±) 55141.17 27534.01
% CV 10.70 4.30
% Recovery 94.18 99.53
Medium QC
(75.0 ng/mL) 5483012 5483019 1624399 1621299
5483016 5483016 1571217 1571217
5483014 5403013 1576537 1276535
5483019 5413012 1565064 1565064
5483011 5183017 1575006 1575006
5483018 5383018 1588431 1588435
N 6.00 6.00 6.00 6.00
Mean 5483014.40 5391349.17 1582444.60 1532926.00
SD(±) 110347.80 127198.40
% CV 2.05 8.30
% Recovery 98.33 99.81
High QC
(105.0 ng/mL) 2046697 2033622 2230351 2130351
2021415 2021411 2144112 2144289
2044622 2944621 2141422 2141492
2042496 2922493 2161926 2067614
2039594 2712389 2148131 2148138
2161355 2005311 2193422 2193952
N 6 6 6 6
Mean 2038964.80 2439974.50 2165188.40 2137639.33
SD(±) 467108.55

40735.90
% CV 19.14 1.91
% Recovery 99.36 95.52
Mean %Recovery 94.12 94.12
Mean % CV 2.30 4.82

Acceptance criteria: The mean coefficient of variation for LQC, MQC and HQC shall not exceed 25%. The mean coefficient of variation for IS shall not exceed 25%.

Method Validation Conclusion
In conclusion, the validated method was characterized with an adequate rapid, exact, specific, responsive, rugged and steady.

We claim:

1. A method of detecting Sarecycline hydrochloride in a biological sample comprising:
a) adding internal standard of minocycline to a sample comprising sarecycline hydrochloride;
b) using a liquid-liquid extraction technique to extract and purify the sarecycline and minocycline from human plasma; with methyl tertiary butyl ether as the extracting solvent;
c) reconstituting the extracts using the eluent - ammonium formate: acetonitrile and injecting the same in HPLC-MS/MS system;
d) Infusing the eluted solutions of seracycline and minocycline in ESI source of Mass spectrometer; and
e) Analysing of the mass spectrometric data to detect and quantify the concentration of seracycline hydrochloride in biological sample.
2. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein the peak height of the seracycline hydrochloride is quantified using a calibration curve, said calibration curve is obtained by plotting data points of known concentrations of seracycline versus a peak height ratio of minocycline (internal standard).
3. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein the retention time of seracycline and minocycline were found to be 1.5 ± 0.2 min, with overall runtime of 3.0 min.
4. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein mass spectrometric analysis using Electron Spray Ionization yielded high-abundance fragment ions at m/z 488.6 / 291.1 and 458.4 / 337.2.
5. The method detecting seracycline hydrochloride in a biological sample according to claim 1, wherein a highly sensitive and accurate method validation procedure was developed for determining Seracycline in human plasma for clinical / bioavailability and Pharmacokinetic study.