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Development of a Highly Sensitive, Efficient, Combination Extraction Method for the Quantitation of Formoterol and Budesonide by UPLC/MS/MSRyan S. Adler, Sherry Liu, Alicia Pietrasiewicz, Spencer J. Carter and Min Meng

Introduction

Method

Method continued

Results and Discussion

Results and Discussion continued

Conclusion

References

• Linearity was established across 2 orders of magnitude at low concentrations (1.00 – 100 pg/mL for formoterol and 3.00 – 300 pg/mL for budesonide) with good accuracy and precision.

• A single solid phase extraction method utilizing two separate elution steps was developed in conjunction with two ~3 minute LC methods, providing the necessary effi ciency required for a high throughput assay.

• UPLC chromatography allowed for short run times while effectively separating endogenous interferences and providing good signal:noise.

Fang X, et al. An LC-MS/MS Method for Simultaneously Determining Formoterol and Budesonide in Human Plasma with Dual HPLC System. The 59th ASMS Conference, Denver, CO, June 2011.

Carter S, et al. Obtaining Sensitive Lower Limits of Quantitation for the Determination of Fluticasone Propionate, Salmeterol, Formoterol, and Budesonide in Human Plasma by LC/MS/MS. Presented at the Respiratory Drug Delivery (RDD) meeting, Orlando, FL, April 2010.

Meng M, et al. A systematic approach for developing a robust LC-MS/MS method for bioanalysis. Bioanalysis (2013); 5(1):91-115.

LC-MS/MSUPLC: Waters Acquity UPLC® systemMass Spec: AB Sciex® QTRAP® 6500 (Life Technologies)Source and ionization: ESI (Positive ion mode)Source Temperature: 500 °CSRM transition: Formoterol 345.0 → 149.1 m/z Budesonide 431.3 → 323.2 m/z Formoterol-d6 351.2 → 155.2 m/z Budesonide-d8 439.3 → 323.2 m/z

FOR FORMOTEROL:

LC Column: Phenyl based stationary phaseLC Flow Rate: 0.4 mL/minLC Mobile phase: A: Acidifi ed ammonium acetate B: MethanolLC program: Gradient with column diversion Run time: 3.5 minutes

FOR BUDESONIDE:

LC Column: C8 based stationary phase.LC Flow Rate: 0.6 mL/minLC Mobile phase: A: Basifi ed ammonium bicarbonate B: Methanol and MeCN mixture LC program: Gradient with column diversion Run time: 3.0 minutes

Sample PreparationMatrix: Human plasma (K2EDTA)

Calibration range: Formoterol 1-100 pg/mL

Budesonide 3-300 pg/mL

Aliquot size: 500 µL

Extraction: Fortifi ed plasma was acidifi ed, loaded onto a mixed mode Strata-XTM-C SPE plate (Phenomenex®), and eluted twice into separate plates; once for formoterol, and once for budesonide. The formoterol eluate was evaporated and reconstituted in an acidic water/MeOH mixture. The budesonide eluate was partially evaporated.

Figure 2. Results of AmoebaTM SPE Extraction Screening Experiment

Figure 3. Phospholipid Management with Forward Flush and Gradient

Figure 5. Chromatograms for Formoterol (LLOQ, Blank, QC0)

Figure 6. Chromatograms for Budesonide (LLOQ, Blank, QC0)

Figure 7. Calibration Curve for Formoterol

Figure 8. Calibration Curve for Budesonide

Table 1. Back-Calculated Concentrations (pg/mL) of Calibration Standards

Table 2. Quality Control Summary of Accuracy and Precision (ANOVA)

Figure 4. Chromatography Comparison for Formoterol using (Isocratic vs. Gradient)

Figure 1. Structures of Formoterol and Budesonide

Formoterol and budesonide (Figure 1) are both used in the management and treatment of asthma and chronic obstructive pulmonary disease (COPD). Formoterol functions as a long acting β2-agonist (LABA) with a 12 hour duration of action. Budesonide, a corticosteroid used as an inhalant to treat asthma, acts as an anti-infl ammatory for the respiratory tract. Dosing of formoterol and budesonide through inhalation leads to low concentrations present in the blood stream. Due to the low concentrations, it is imperative to develop a highly sensitive LC/MS/MS assay.

In previous publications, formoterol and budesonide were either quantifi ed separately at 1.00 pg/mL and 10.0 pg/mL respectively [S. Carter, 2010], requiring a 500 µL aliquot for each assay, or quantifi ed simultaneously at 0.400 pg/mL and 10 pg/mL, respectively, using complicated dual HPLC systems [X Fang, et al, 2010]. In this presentation, we report a one-extraction and two-injection method to quantify formoterol and budesonide at 1.00 pg/mL and 3.00 pg/mL. To the best of our knowledge, this is the lowest LLOQ ever reported for the quantitation of budesonide.

Method Development

1. OPTIMIZATION OF SAMPLE CLEAN-UP Because the main goal of this assay was to develop a sensitive

assay with low pg/mL limits of quanitiation, a large aliquot volume and a thorough sample clean-up were deemed to be required. Additionally, manual extraction with a cartridge-based format was not considered, as it is not suitable for high throughput production. Therefore, the focus was to optimize the SPE extraction procedure with a 96-well plate format. A SPE screening experiment was conducted according to AmoebaTM screening protocols (LabCorp) [Meng, et al, 2013] and budesonide demonstrated similar recoveries under all tested conditions (Figure 2). Likewise, formoterol showed similar recoveries except when using Strata-X-C. While no recovery was observed under neutral elution conditions, good recovery was observed with a basic elution. This feature made it possible to separate Formoterol and Budesonide in a single extraction, i.e. elute budesonide fi rst under neutral conditions, then elute formoteral from the same sample plate under basic conditions.

Method Validation

This method was fully validated under GLP conditions. Validation experiments included intra- and inter-day precision and accuracy, selectivity/specifi city, hemolysis, matrix effects, interference and various stability tests. The method passed all validation tests with excellent linearity and great precision and accuracy.

LINEARITY: Excellent accuracy and precision were demonstrated throughout with single digit % CV and % bias for calibration standards in all three validation runs using linear 1/x2 regression (Table 1). The average R2 values were: 0.9982 (formoterol) and 0.9976 (budesonide). See Figures 5 & 6 for representative LLOQ, control blank and QC0 chromatograms and see Figures 7 & 8 for representative calibration curves.

Note: Blue bar = Formoterol; Red bar = Budesonide

2. OPTIMIZATION OF LC CONDITIONS Virtually all types of LC columns (i.e. C8, C18, Phenyl, biphenyl, and

PFP) were able to successfully retain formoterol and budesonide with good peak shape when using neat solutions at high concentrations. However, with extracted plasma samples, the background varied from column to column, especially at the LLOQ level. Phospholipids were monitored throughout the LC development, and were adequately managed by forward fl ushing the column after each injection (Figure 3). Additionally, modifi cations to the LC gradient had great impact on the signal to noise (S/N) of the LLOQ in extracted plasma samples as well (Figure 4).

FORMOTEROL

Analytical Run

Number

1.00(pg/mL)

2.00(pg/mL)

5.00(pg/mL)

10.0(pg/mL)

25.0 (pg/mL)

50.0(pg/mL)

90.0(pg/mL)

100 (pg/mL)

1 0.961 1.98 5.03 10.1 25.0 48.8 86.3 98.7

1.03 2.03 5.12 10.4 25.6 49.5 89.1 99.4

4 0.973 2.07 5.14 10.3 24.6 48.7 84.4 100

0.987 2.01 5.29 10.3 24.7 49.5 86.2 102

7 0.886 1.93 5.08 10.1 25.2 49.5 88.8 97.3

1.12 1.99 5.14 10.3 25.2 49.6 88.8 101

Mean 0.993 2.00 5.13 10.3 25.1 49.3 87.3 99.7

S.D. 0.078 0.0475 0.0876 0.122 0.367 0.403 1.92 1.67

%CV 7.9 2.4 1.7 1.2 1.5 0.8 2.2 1.7

%Bias -0.7 0 2.6 3 0.4 -1.4 -3 -0.3

n 6 6 6 6 6 6 6 6

BUDESONIDE

Analytical Run

Number

3.00(pg/mL)

6.00 (pg/mL)

15.0(pg/mL)

30.0(pg/mL)

75.0 (pg/mL)

150(pg/mL)

270(pg/mL)

300(pg/mL)

1 2.77 5.41 15.1 29.6 74.1 147 266 297

3.30 6.22 15.4 30.3 78.0 154 267 305

4 3.18 5.57 15.9 31.1 71.9 149 257 300

2.87 5.99 15.4 30.9 73.8 151 267 305

7 3.02 5.93 16.1 30.5 73.2 144 263 290

2.91 5.91 16.3 31.0 77.1 149 261 292

Mean 3.01 5.84 15.7 30.6 74.7 149 264 298

S.D. 0.2 0.296 0.469 0.565 2.36 3.41 3.99 6.37

%CV 6.6 5.1 3 1.8 3.2 2.3 1.5 2.1

%Bias 0.3 -2.7 4.7 2.0 -0.4 -0.7 -2.2 -0.7

n 6 6 6 6 6 6 6 6

* - No signifi cant additional variation was observed as a result

FORMOTEROL

Nominal Conc. LLOQ(1.00 pg/mL)

Low(3.00 pg/mL)

Low-Medium

(15.0 pg/mL)

Medium(40.0 pg/mL)

High(80.0 pg/mL)

Mean Observed Conc. 0.95 2.89 13.7 39.1 76.9

%Bias -5.0 -3.7 -8.7 -2.3 -3.9

Between Run Precision (%CV) 0.0* 0.6 0.1 0.0* 1.3

Within Run Precision (%CV) 5.6 3.3 0.9 2.0 1.7

Total Variation (%CV) 5.5 3.4 0.9 1.8 2.2

n 18 18 18 18 18

Number of Runs 3 3 3 3 3

BUDESONIDE

Nominal Conc. LLOQ(3.00 pg/mL)

Low(9.00 pg/mL)

Low-Medium

(45.0 pg/mL)

Medium(120 pg/mL)

High(240 pg/mL)

Mean Observed Conc. 2.84 8.76 42.5 122 239

%Bias -5.3 -2.7 -5.6 1.7 -0.4

Between Run Precision (%CV) 0.0* 0.0* 0.0* 1.1 0.6

Within Run Precision (%CV) 8.2 3.7 1.9 1.9 1.7

Total Variation (%CV) 7.7 3.6 1.9 2.2 1.8

n 18 18 18 18 18

Number of Runs 3 3 3 3 3

ACCURACY/PRECISION: Excellent accuracy and precision was dem-onstrated throughout with single digit % CV and % bias for QCs (n=6) at LLOQ, Low, Low-Medium, Medium and High concentrations dur-ing three validation runs (Table 2).

SELECTIVITY: Selectivity was demonstrated in six individual sources of human plasma K2EDTA in both control blanks and at the LLOQ concentration (data not shown).

ABILITY TO DILUTE: The ability to dilute was evaluated and met ac-ceptance above the ULOQ at DF=10 (data not shown).

EXTRACTION RECOVERY: Extraction recovery was evaluated (n=6) and met acceptance at the Low, Medium and High concentrations for both analytes and at the working concentration for the internal standards. The overall recovery was 66.6-71.8% for formoterol and 22.0-24.5% for budesonide.

MATRIX FACTOR: Matrix factor was evaluated (n=6) and met acceptance for both analytes at the Medium QC concentration and at the working concentration for the internal standards. The non-normalized and normalized matrix factors for formoterol and budesonide were 0.87/0.93 and 1.00/1.05, respectively.

CARRYOVER: Carryover was evaluated and met acceptance in each validation and subsequent sample analysis run. No carryover was present in control blank samples following the highest standard calibrator.

HEMOLYSIS: Hemolysis was evaluated (0% hemolysis, 0.5% hemolysis and 2.0% hemolysis) and met acceptance for both analytes at the Low concentration.

STABILITY: The following stability assessments were performed and met acceptance: Freeze/thaw stability (4 cycles) Bench-top matrix stability (6 hours) Short-term matrix stability (32 days at -20 °C) Whole blood stability (up to 2 hours at ambient temperature)

REINJECTION REPRODUCIBILITY: Reinjection reproducibility was established for 165.6 hours for formoterol and 177.2 hours for budesonide at RT (data not shown)

EXTRACT STABILITY: Extract stability was evaluated and established for up to 70 hours at RT (data not shown)

BATCH SIZE: Batch size was evaluated and established for up to 192 samples (data not shown)