DESC:TECHNICAL FIELD OF THE INVENTION
The invention relates to a high performance liquid chromatography-mass spectrometry (HPLC-MS) method for quantitation of 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine), 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2) levels in a plasma sample.

BACKGROUND OF THE INVENTION

Human insulin is a peptide hormone composed of 51 amino acids, and has a molecular weight of 5808 daltons [Da]. It is a hetero dimer of two peptide chains connected by disulfide bonds that is secreted by beta cells of pancreas, and is central to regulating carbohydrate and fat metabolism in the body. Insulin disturbance can cause diabetes mellitus, a condition in which the pancreas no longer produces enough insulin or cells stop responding to the insulin that is produced, so that glucose in the blood cannot be absorbed into the cells.
Animal insulin, including Porcine and Bovine insulin, has been used clinically for the treatment of diabetes. However, biosynthetic/recombinant human insulin is preferred because side effects are generally less common. Biotechnology started introduction of reengineered human insulin in the late 1980s, and this insulin can achieve somewhat different absorption or duration of action characteristics. For example, NovoRapid® and Apidra® are rapid-acting analogues, and Lantus® and Levemir® are the types of long-acting insulin analogues. During the drug development and clinical study, the fundamental information including insulin presence, concentration, metabolism of insulin and its related compounds collected needs to be answered to understand medical treatments for patients suffering from different types of diabetes under individual conditions.

Insulin glargine design followed the physiology of human insulin formation in b-cells in which 31B-Arg-32BArg- human insulin is a final intermediate during the processing from pro insulin to human insulin. Although unmodified 31B-Arg-32B-Arg-human insulin failed subcutaneously despite being fully active intravenously, substitution of 21A-Asp for 21A-Gly rendered the molecule both chemically stable and fully active subcutaneously without substantial alterations in receptor affinities. Soluble at acidic pH, glargine precipitates amorphously upon subcutaneous injection and becomes subject to enzymatic maturation into 21A-Gly-human insulin upon slow release from the depot. As a result, glargine exhibits a nearly flat action profile and duration beyond 24 hours after multiple dosing in subjects with type 1 and type 2 diabetes. Glargine is preferred to human NPH insulin because it reduces the risk of hypoglycaemia, primarily nocturnal.

In vitro studies have indicated that glargine has greater binding affinity for the IGF-1 receptor (IGF-1R) and greater potency on DNA synthesis (so-called mitogenic effects) compared with human insulin, at least in malignant cell lines expressing primarily IGF-1R, not insulin receptors (IR). However, the in vitro data are not directly applicable in vivo in humans; the natural precursor 31B-Arg-32B-Arg-human insulin shows even greater IGF-1R affinity than glargine. In addition, it is presently proposed that the mitogenic potential of insulin analogs is mediated primarily via IR, not IGF-1R. Nevertheless, the safety of glargine in humans has been questioned based on in vitro experiments, even though glargine does not promote tumour growth in vivo in animals, in contrast to the insulin analog X10 (10B-Asp-human insulin), which presents with greater affinity for both IR and IGF-1R. Some controversial registry studies have suggested a possible greater cancer risk in humans using glargine versus non glargine insulin.

After subcutaneous injection, glargine undergoes an enzymatic removal of the basic arginine pair at positions 31B and 32B to yield 21A-Gly-human insulin (metabolite M1), analogous to pro hormone activation, with some further loss of threonine to 21A-Gly-des-30B-Thr-human insulin (metabolite M2). Both M1 and M2 exhibit lower affinity for IGF-1R and lower mitogenic potential in vitro compared with glargine, and even with human insulin, while fully retaining its metabolic properties. Thus, it is understood that most, if not all, of the glargine injected subcutaneously in humans is rapidly transformed to M1 and partly further to M2, resulting in minimal, if any, plasma exposure to parent glargine. Yet, because of technical constraints, the in vivo quantification of glargine metabolism to M1 and M2 in humans has so far been limited and of uncertain interpretation.

US Patent Publication No. 20150087073 A1 discloses methods for quantifying polypeptides using mass spectrometery. Bolli et al Diabetes care Dec 2012 discloses the study to quantitate plasma concentrations of glargine, M1, and M2 after subcutaneous injection of glargine in male type 1 diabetic subjects. Li et al, J Anal Bioanal Tech 2013 discloses study of quantitation of insulin glargine metabolites M1 and M2 using bovine insulin as internal standard with Triple Quad 6500 and triple TOF 5600 LC-MS/MS systems in a dog toxicokinetics study.

The quantitative analysis of peptides and proteins using LC-MS/MS depends on the use of appropriate internal standard spiked prior to sample processing stage. The first reason, internal standard is spiked prior to sample clean-up is to ensure that internal standard and test analyte receive same treatment and thus it confirms that test analyte is not degraded/lost during sample treatment. Another reason for introduction of internal standard is use of electrospray ionisation mode for introduction of ionized analytes in the mass spectrometer for quantitation. The electrospray ionization is responsible for ionization of analytes from solution state to gaseous state. During electrospray ionization, the application voltage to spraying tip generates small droplets; from these droplets the solvent evaporates to generate ions in gaseous state. The ions in the gaseous state get detected and quantitated in mass spectrometer. This ionization process is dependent on multiple factors such as voltage stability, solvent composition (higher organic solvent content favours ionization as it evaporates faster) and salts deposited on the spraying tip. In order to ensure that the ionization of the internal standard and analyte occurs at the same solvent composition, they need to elute at same retention time or close to each other during chromatography.

Inventors of present application surprisingly found that use of 31B-Arg-32B-Arg-human insulin (B32-insulin) as internal standard for Insulin Glargine and des-30B-Thr-human insulin (B29-insulin) as internal standard for 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2). Inventors used understanding about the various sequence variants available of insulin and Insulin Glargine to select internal standards for insulin glargine and its metabolites M1 & M2. The amino acid sequence of B32 Insulin is similar to Insulin Glargine except position A21 (i.e.B32 Insulin has Asparagine at 21A position, whereas Insulin Glargine has Glycine at 21A position). Both species elute at same retention time due to similar amino acid sequence and thus help in accurate quantitation. The M2 has similar amino acid sequence to that of B29 insulin except position 21A (B29 Insulin has Asparagine at 21A position, whereas M2 has Glycine at 21A position). The amino acid sequence of M1 metabolite has additional threonine amino acid which does not contribute significantly towards the change in the retention time compared to M2. Thus M1 and M2 elute very closely and a single internal standard is used for their quantitation.
So it is an objective of the present invention to develop an analytical method for quantitation of 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine), 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2) levels in a plasma sample using 31B-Arg-32B-Arg-human insulin (B32-insulin) and des-30B-Thr-human insulin (B29-insulin) as internal standards.

The application of internal standard with close elution chromatographic pattern and with different mass ensures that the ionization of the analyte and internal standard specified occurs at same solvent composition and thus provides superior and accurate quantitation.

SUMMARY OF THE INVENTION

In one general aspect of the invention, there is provided a High performance liquid chromatography-Mass spectrometry (HPLC-MS) method for quantitation of 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine), 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2) levels in a plasma sample.

In another aspect of the invention, there is provided a high performance liquid chromatography-Mass spectrometry (HPLC-MS) method for quantitation of 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine), 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2) levels in a plasma sample, said method comprising the steps of:
i. preparation of 21A-Gly-31B-Arg-32B-Arg-human insulin solution, 21A-Gly-human insulin solution and 21A-Gly-des-30B-Thr-human insulin solution in a suitable solvent for compatibility with plasma samples;
ii. preparation of 31B-Arg-32B-Arg-human insulin (B32-insulin) solution and des-30B-Thr-human insulin (B29-insulin) solution in a suitable solvent to be used as internal standards;
iii. spiking of internal standard mixture prepared in step (ii) in plasma sample prepared in step (i) to quantitate 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin Glargine), 21A-Gly-human insulin (insulin Glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin Glargine metabolite M2)
iv. injecting the samples of step (iii) on a suitable reverse phase HPLC column maintaining appropriate temperature and gradient of mobile phase; and
v. eluting 21A-Gly-31B-Arg-32B-Arg-human insulin, 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin and the internal standards from reverse phase HPLC column to a suitable detector;

In another aspect of the invention, wherein 31B-Arg-32B-Arg-human insulin (B32-insulin) is used as internal standard to quantitate 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin Glargine) as their retention times are similar.

In another aspect of the invention, wherein des-30B-Thr-human insulin (B29-insulin) is used as internal standard to quantitate 21A-Gly-human insulin (insulin Glargine metabolite M1) as their retention times are similar.

In another aspect of the invention, wherein des-30B-Thr-human insulin (B29-insulin) is used as internal standard to quantitate 21A-Gly-des-30B-Thr-human insulin (insulin Glargine metabolite M2) as their retention times are similar.

In another aspect, the plasma sample is from source of human or any mammalian species plasma sample.

In another aspect, the suitable solvent for preparation of the samples and the internal standards is a diluent. In another aspect, the diluent comprises 0.1 %v/v formic acid in 1.0 %v/v bovine serum albumin solution in water.

In another aspect, the reverse phase HPLC column has a C18 solid phase with a median particle size of 1.7µ and a median particle size pore of 100x2.1 mm. In another aspect, the temperature of column is about 60°C.
In another aspect, the mobile phase is acetonitrile (A): 0.02%v/v formic acid in water (B).

In another aspect, the injecting volume of samples is about 10µl. In another aspect, the eluting run time of samples is about 12 minutes.

In another aspect, the detector is mass spectrometer.

In another aspect, the relative retention time of internal standard 31B-Arg-32B-Arg-human insulin is 4.68 minutes. In another aspect, the relative retention time of 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin is 4.76 minutes and 4.75 minutes respectively. In another aspect, the relative retention time of internal standard des-30B-Thr-human insulin is 3.17 minutes. In another aspect, the relative retention time of 21A-Gly-31B-Arg-32B-Arg-human insulin is 3.25 minutes.

In another aspect, the method has about 0.1 ng/ml of a lower limit of detection for 21A-Gly-31B-Arg-32B-Arg-human insulin. In another aspect, the method has about 0.1 ng/ml of a lower limit of detection for 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the representative calibration curve for recombinant insulin glargine (GLA).
Figure 2 is the representative chromatogram of blank sample.
Figure 3 is the representative chromatogram of LLOQ.
Figure 4 is the representative chromatogram of ULOQ.

DETAILED DESCRIPTION OF THE INVENTION

While the invention has been described in term of its specific embodiments, certain modification and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the invention.

In one general embodiment of the invention, there is provided a high performance liquid chromatography-Mass spectrometry (HPLC-MS) method for quantitation of 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine), 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2) levels in a plasma sample.

In one embodiment of the invention, there is provided a high performance liquid chromatography-Mass spectrometry (HPLC-MS) method for quantitation of 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine), 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2) levels in a plasma sample, said method comprising the steps of:
i. preparation of 21A-Gly-31B-Arg-32B-Arg-human insulin solution, 21A-Gly-human insulin solution and 21A-Gly-des-30B-Thr-human insulin solution in a suitable solvent as plasma samples;
ii. preparation of 31B-Arg-32B-Arg-human insulin (B32-insulin) solution and des-30B-Thr-human insulin (B29-insulin) solution in a suitable solvent to be used as internal standards;
iii. spiking of internal standard mixture prepared in step (ii) in plasma sample prepared in step (i) to quantitate 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin Glargine), 21A-Gly-human insulin (insulin Glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin Glargine metabolite M2)
iv. injecting the samples of step (iii) on a suitable reverse phase HPLC column maintaining appropriate temperature and gradient of mobile phase; and
v. eluting 21A-Gly-31B-Arg-32B-Arg-human insulin, 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin and the internal standards from reverse phase HPLC column to a suitable detector;

In another embodiment of the invention, wherein 31B-Arg-32B-Arg-human insulin (B32-insulin) is used as internal standard to quantitate 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin Glargine) as their retention times are similar.

In another embodiment of the invention, wherein des-30B-Thr-human insulin (B29-insulin) is used as internal standard to quantitate 21A-Gly-human insulin (insulin Glargine metabolite M1) as their retention times are similar.

In another embodiment of the invention, wherein des-30B-Thr-human insulin (B29-insulin) is used as internal standard to quantitate 21A-Gly-des-30B-Thr-human insulin (insulin Glargine metabolite M2) as their retention times are similar.

In another embodiment, the plasma sample is from source of human or any mammalian species plasma sample.

In another embodiment, blank plasma is harvested in K3EDTA anticoagulant for the preparation of plasma calibration standards.

In another embodiment, the plasma sample is human K3EDTA plasma.

In another embodiment, the suitable solvent for preparation of the samples and the internal standards is a diluent. In another aspect, the diluent comprises 0.1 %v/v formic acid in 1.0 %v/v bovine serum albumin solution in water.

In another embodiment, the reverse phase HPLC column has a C18 solid phase with a median particle size of 1.7µ and a median particle size pore of 100x2.1 mm. In another embodiment, the temperature of column is about 60°C or is about 50°C or about 70°C. In another embodiment, the temperature of column is about 60°C.

In another embodiment, the mobile phase is acetonitrile (A): formic acid in water (B).
In another embodiment, the mobile phase is acetonitrile (A): 0.02%v/v formic acid in water (B). In another embodiment, the mobile phase is in gradient mode.

In another embodiment, the injecting volume of samples is about 10µl. In another embodiment, the eluting run time of samples is about 12 minutes.

In another embodiment, the detector is mass spectrometer.

In another embodiment, the relative retention time of internal standard 31B-Arg-32B-Arg-human insulin is 4.68 minutes. In another embodiment, the relative retention time of 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin is 4.76 minutes and 4.75 minutes respectively. In another embodiment, the relative retention time of internal standard des-30B-Thr-human insulin is 3.17 minutes. In another embodiment, the relative retention time of 21A-Gly-31B-Arg-32B-Arg-human insulin is 3.25 minutes.

In another embodiment, the method has about 0.1 ng/ml of a lower limit of detection for 21A-Gly-31B-Arg-32B-Arg-human insulin. In another aspect, the method has about 0.1 ng/ml of a lower limit of detection for 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin.
EXAMPLES
EXAMPLE 1: Preparation of Solutions
1.1 Bovine serum albumin solution (BSA)
50.00 mg of bovine serum albumin was weighed accurately in polypropylene tube and dissolved in 4.500mL of water. 0.050mL of acetic acid, 0.020mL of ortho phosphoric acid and 0.430mL of water were added into above solution and mixed well with 15 second vortexing.
1.2. Diluent [0.1 %v/v formic acid in (1.0 %v/v bovine serum albumin solution in water)]
2.000mL of bovine serum albumin solution was transferred accurately into a glass bottle containing198.00 mL of water and mixed well with 15 second vortexing. 0.200mL of formic acid was transferred accurately into a glass bottle containing 199.80mL of above solution and mixed well with 15 second vortexing.
1.3. Buffer for mobile phase [0.02%v/v formic acid in water] (MBUF)
0.100 mL of formic acid was transferred accurately into a glass bottle containing 499.90mL of water. The solution was sonicated to mix well (5 minutes 25 ?C).
1.4. Buffering agent (5.0%v/v ammonia solution in water) (BUF)
10.00 mL of ammonia solution was transferred accurately into a glass bottle containing 190.00mL of water. The solution was sonicated to mix well (5 minutes 25 ?C).
1.5. Washing solution [0.05%v/v ammonia solution in water] (WAS)
0.250mL of ammonia solution was transferred accurately into a glass bottle containing 499.75mL of water. The solution was sonicated to mix well (5 minutes 25 ?C).
1.6. Elution solution [0.5%v/v acetic acid in methanol] (ES)
2.500mL of acetic acid was transferred accurately into a glass bottle containing 497.50mL of methanol. The solution was sonicated to mix well (5 minutes 25 ?C).
EXAMPLE 2: Preparation of Stock Solutions
2.1 Insulin glargine main stock solutions (Analyte)
(a) Main stock solution A (0.1000 mg/mL)
1.000 mg of recombinant insulin glargine working standard (manufactured by Wockhardt Ltd) was weighed accurately and transferred into a 10.00 mL volumetric flask containing 5.000 mL of diluent and gently vortexed to dissolve and diluted up to the mark with diluent.
(b) Main stock solution B (0.1000mg/mL)
1.000mg of recombinant insulin glargine working standard (manufactured by Wockhardt Ltd) was weighed accurately and transferred into a 10.00 mL volumetric flask containing 5.000 mL of diluent and gently vortexed to dissolve and diluted up to the mark with diluent.
2.2 Insulin glargine M1 main stock solutions (Analyte)
(c) Main stock solution A (0.1000 mg/mL)
1.000 mg of recombinant insulin glargine M1 working standard (manufactured by Wockhardt Ltd) was weighed accurately and transferred into a 10.00 mL volumetric flask containing 5.000 mL of diluent and vortexed for 15 second to dissolve and diluted up to the mark with diluent.
(d) Main stock solution B (0.1000 mg/mL)
1.000 mg of Insulin glargine M1 working/reference standard was weighed accurately and transfer into a 10.00 mL volumetric flask containing 5.000 mL of diluent and gently vortexed to dissolve and diluted up to the mark with diluent.
2.3 Insulin glargine M2 main stock solutions (Analyte)
(e) Main stock solution A (0.1000 mg/mL)
1.000 mg of Recombinant Insulin glargine M2 working standard (manufactured by Wockhardt Ltd) was weighed accurately and transferred into a 10.00 mL volumetric flask containing 5.000 mL of diluent and gently vortexed to dissolve and diluted up to the mark with diluent.
(f) Main stock solution B (0.1000 mg/mL)
1.000 mg of Insulin glargine M2 working/reference standard was weighed accurately and transferred into a 10.00 mL volumetric flask containing 5.000 mL of diluent and gently vortexed to dissolve and diluted up to the mark with diluent.
2.4 Mixed intermediate stock solutions (Insulin glargine, M1 & M2)
(g) Mixed intermediate stock solution A (2000.000 ng/mL)
0.100 mL of solution prepared in example 2.1 (a), 0.100 mL of solution prepared in example 2.2 (c) and 0.100mL of solution prepared in example 2.3 (e) were transferred accurately into a 5.00 mL volumetric flask and diluted up to the mark with diluent.
(h) Mixed intermediate stock solution B (2000.000nglmL)
0.100 mL of solution prepared in example 2.1 (b), 0.100 mL of solution prepared in example 2.2 (d) and 0.100 mL of solution prepared in example 2.3 (f) were transferred accurately into a 5.00 mL volumetric flask and diluted up to the mark with diluent.
2.5 B-29&B-32 Insulin main stock solutions (Internal standards)
(i) B-29 Insulin main stock solution (0.1000 mg/mL)
1.000 mg of Recombinant B-29 Insulin working standard (manufactured by Wockhardt Ltd) was weighed accurately and transferred into a 10.00 mL volumetric flask containing 5.000 mL of diluent and gently vortexed to dissolve and diluted up to the mark with diluent.
(j) B-32 Insulin main stock solution (0.1000 mg/mL)
1.000 mg of Recombinant B-32 Insulin working standard (manufactured by Wockhardt Ltd) was weighed accurately and transferred into a 10.00mL volumetric flask containing 5.000mL of diluent and gently vortexed to dissolve and diluted up to the mark with diluent.
2.6 Mixed internal standard working stock solution (80.000 ng/mL of B-29 and (k))
(k) 300.000ng/mL of B-32)
Transferred accurately 0.008 mL of solution prepared in example 2.5 (i) and 0.030 mL of solution prepared in example 2.4 (g) into a ria tube and added 9.962 mL of diluent.
EXAMPLE 3: Sample Preparation
The analyte free matrix was retrieved from the deep freezer and allowed for thawing at room temperature and vortexed for 15 second before processing.
3.1 Preparation of blank samples
0.015mL of diluent was added to 0.285 mL of analyte free matrix in a micro-centrifuge tube and vortexed to mix. 0.015 mL of diluent was added during sample treatment.
3.2 Preparation of zero samples
0.015 mL of diluent was added to 0.285 mL of analyte free matrix in a micro-centrifuge tube and vortexed to mix. 0.015 mL of mixed internal standard working stock solution was added during sample treatment.

3.3 Preparation of Insulin Glargine, M1 and M2 calibration standards and quality control samples
The mixed intermediate stock solutions were transferred and diluent was added to make the final volume. Mixed intermediate stocks prepared in example 2.4 (g) was used for calibration standard preparation and standard stock solution preparation. Mixed intermediate stock prepared in example 2.4 (h) was used for quality control samples preparation.

EXAMPLE 4: Solid Phase Extraction (SPE) and Mass Spectrometry LC-MS/MS
i. The samples were retrieved from deep freezer and allowed for thawing in ice cold water bath.
ii. Each sample was gently vortexed for 15 seconds adequately using a vortexer before processing.
iii. 0.300 mL sample was transferred into microcentrifuge tube and 0.015 mL of internal standard working stock solution was added, except in blank sample and gently vortexed to mix.
iv. 0.300 mL of buffering agent was added and gently vortexed to mix.
v. The above plasma mixture was loaded on previously conditioned (with 1.000 mL of methanol and 1.000 mL of water twice, sequentially) Oasis HLB lcc/30mg cartridges.
vi. The cartridges were washed thrice with 1.000 mL of washing solution (0.05%v/v ammonia solution in water).
vii. The cartridges were eluted twice with 0.500mL of elution solution (0.5%v/v Acetic acid in Methanol) in ria tube.
viii. The cartridges were evaporated to dryness under stream of nitrogen at 30°C
ix. The analyte residue was reconstituted with 0.150 mL of reconstitution solution (0.05%v/v Bovine serum albumin solution).
x. The samples were injected 10.0µL into LC-MS/MS.

4.1 Chromatographic Conditions
Table 1
Chromatographic mode Reverse Phase
Isocratic/gradient mode Gradient
Weak wash solution 0.1%v/v Formic acid in Water
Strong wash solution 0.1 %v/v Formic acid in [Acetonitrile: Water: Methanol: Isopropanol (25:25 :25:25%v/v)]
Weak wash volume 2000µL
Strong wash volume 2000µL
Target sample temp. (°C) 10
Injection volume 10µL
Column Acquity UPLC C18 2.1 *100mm,l.7um
Mobile phase Acetonitrile (A): 0.02%v/v Formic acid in Water (B)
Target column temperature (°C) 60
Run time (min) 12
Retention time (min) Recombinant insulin glargine (3.25); Recombinant insulin B-32 (3.17); Recombinant insulin glargine M1 (4.76); Recombinant insulin B-29 (4.68); Recombinant insulin glargine M2 (4.75)

4.2 Detector Parameters: Mass spectrometer
Table 2
Ionization mode ESI+VE
Capillary (kV) 3.90
Cone(V) 21
Source offset(V) 50
Source temperature CCC) 150
Desolvation temp (0C) 500
Cone gas flow(L/Hr) 150
Desolvation gas flow (L/Hr) 1000
Collision gas flow(mLimin) 0.15
Nebuliser gas flow (Bar) 7.00
Data Type Continuum
Scan Type MRM ((Multiple Reaction Monitoring)
Divert Valve Setting NA

Table 3: Detector Parameters
Name Charge state Prnt(Da) Dau(Da)
Insulin glargine 7+ 867.10 984.14
M1 6+ 959.41 1131.26
M2 6+ 942.54 1098.20
B-29 6+ 952.10 1115.86
B-32 6+ 1021.03 1190.96

Where Prnt: Parent ion and Dau: Daughter ion being monitored

4.3 Quantitation
1. The quantitation was performed using Mass Lynx. Software.
2. Chromatogram of analytes and internal standards were constructed and area under the peak for quantitation was calculated.
3. Response was calculated i.e. ratio of area under the peak of analytes to area under the peak of respective internal standards.
4. Response was plotted against the increasing concentrations of analytes to construct the calibration curve.
Calculation
Concentration of analyte(s) was calculated using equation of regression line,
y = mx + c
Where,
y = response i.e. ratio of area under the peak of analytes to area under the peak of respective internal standards
m = slope of calibration curve
x =concentration in ng/mL of analyte(s)
c = Intercept of calibration curve

Table 4: Readings for Calibration Curve
Concentration of analyte (x) Analyte area (a) Internal standard area (b) Response x=a/b
Blank 10524
0.201 1565 9576 0.163429407
0.403 2866 10554 0.271555808
0.604 4150 10149 0.408907282
1.208 8815 10043 0.877725779
2.417 18044 9470 1.905385428
4.834 38597 10260 3.761890838
7.25 61277 9793 6.257224548
9.667 69180 9464 7.309805579
12.084 88150 8640 10.2025463

Table 5: Result
Analyte RT (Minutes) Area IS Area
21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine) 3.25 1565 9576
21A-Gly-human insulin
(insulin glargine metabolite M1) 4.76 475 13019
21A-Gly-des-30B-Thr-human insulin
(insulin glargine metabolite M2) 4.75 596 13019
31B-Arg-32B-Arg-human insulin
(B32-insulin) 3.17 9576 -
des-30B-Thr-human insulin
(B29-insulin) 4.68 13109 -

,CLAIMS:We claim:

1. A high performance liquid chromatography-Mass spectrometry (HPLC-MS) method for quantitation of 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin glargine), 21A-Gly-human insulin (insulin glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin glargine metabolite M2) levels in a plasma sample, said method comprising the steps of:
i. preparation of 21A-Gly-31B-Arg-32B-Arg-human insulin solution, 21A-Gly-human insulin solution and 21A-Gly-des-30B-Thr-human insulin solution in a suitable solvent as plasma samples;
ii. preparation of 31B-Arg-32B-Arg-human insulin (B32-insulin) solution and des-30B-Thr-human insulin (B29-insulin) solution in a suitable solvent to be used as internal standards;
iii. spiking of internal standard mixture prepared in step (ii) in plasma sample prepared in step (i) to quantitate 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin Glargine), 21A-Gly-human insulin (insulin Glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin Glargine metabolite M2)
iv. injecting the samples of step (iii) on a suitable reverse phase HPLC column maintaining appropriate temperature and gradient of mobile phase; and
v. eluting 21A-Gly-31B-Arg-32B-Arg-human insulin, 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin and the internal standards from reverse phase HPLC column to a suitable detector;

2. The method of claim 1, wherein 31B-Arg-32B-Arg-human insulin (B32-insulin) is used as internal standard to quantitate 21A-Gly-31B-Arg-32B-Arg-human insulin (insulin Glargine) as their retention times are similar.

3. The method of claim 1, wherein des-30B-Thr-human insulin (B29-insulin) is used as internal standard to quantitate 21A-Gly-human insulin (insulin Glargine metabolite M1) and 21A-Gly-des-30B-Thr-human insulin (insulin Glargine metabolite M2) as their retention times are similar.

4. The method of claim 1, wherein the plasma sample is human or any mammalian species plasma sample.

5. The method of claim 1, wherein the suitable solvent for preparation of the samples and the internal standards is a diluent, wherein diluent comprises 0.1 %v/v formic acid in 1.0 %v/v bovine serum albumin solution in water

6. The method of claim 1, wherein the mobile phase is acetonitrile (A): 0.02%v/v formic acid in water (B).

7. The method of claim 1, wherein the relative retention time of internal standard 31B-Arg-32B-Arg-human insulin is 4.68 minutes.

8. The method of claim 1, wherein the relative retention time of 21A-Gly-human insulin and 21A-Gly-des-30B-Thr-human insulin is 4.76 minutes and 4.75 minutes respectively.

9. The method of claim 1, wherein the relative retention time of internal standard des-30B-Thr-human insulin is 3.17 minutes.

10. The method of claim 1, wherein the relative retention time of 21A-Gly-31B-Arg-32B-Arg-human insulin is 3.25 minutes.