overcomingextractionchallengesofabisphosphonate ...€¦ · bioanalytical department at comac...

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Overcoming extraction challenges of a bisphosphonatedrug from human plasma utilizing an LLE with phaseseparation approach

Snezhana TraykovaBioanalytical department at Comac Medical Ltd.EBF Young Scientists Symposium – March 2018

Introduction- Ibandronic Acid (IBA)

S. Traykova, EBF Young Scientists Symposium – March 2018 2

• Nitrogen-containing bisphosphonate• Inhibits osteoclast-mediated bone resorption• FPS inhibitor• Hydroxylapatite antagonist

Fig.1 Ibandronic acid 3D chemical structure (Image by PubChem NIH)


• Highly polar• Highly hydrophilic• Poorly adsorbed• Good protein binding• Low matrix concentrations

Chemical Properties

LC-MS-MSmethod development complications


Analyte extraction from matrix

LC-MS MS analysis

Analyte quantification

The Strategy

√

√

√

Why Liquid-Liquid Extraction?


VS

LLE

Solid Phase


The Extraction Techniques

pH adjustment and PP with HCL and K2HPO4

Extraction with Ethyl Acetate

Acidification of the subnatant layer

Purification by evaporation

Derivatization with TMSD

Incubation

2nd purification

Reconstitution with 50% MeOH

Extraction with Ethyl Acetate/ TBME

Direct evaporation of subnatant/supernatant layer

Reconstitution with 50% MeOH

VSA typical liquid-liquid extractionLLE with phase separation

Overcoming the challenge


The described extraction utilizes a combination of these techniques:

• Acidification• Protein precipitation• Phase separation• Derivatization• Reconstitution

Extraction advantages

Increase in recovery Increase in stability Reproducible results Results reliability

The Data Says It All- Low Extraction Yields


Trial extraction procedure- representative chromatogram of IBA at a concentration of 2.5150 µg/L

min0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40

%

-15

85

MRM of 2 channels,ES+379.25 > 117.17

CS02_0922_01 Smooth(Mn,8x2)

3.447e+004d3-IBA;1.01;2732;31049;152

0.06

min

%

-15

85


CS02_0922_01 Smooth(Mn,8x2)

4.009e+0031.32IBA;1.00;202;2213;140.08

0.32 0.46

The Data Says It All- Successful Extraction From Plasma


Final extraction procedure - representative chromatogram of IBA at a concentration of 2.5150 µg/L

min1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

-15

85


AR_07_CS02 Smooth(Mn,8x2)589

1.291e+005d3-IBA;1.54;17602;125828;918

1.25

min

%

-15

85



2.814e+004IBA;1.59;3636;25198;166

1.26

2.62

The Data Says It All- Low Extraction Yields


Trial extraction procedure- representative chromatogram of IBA at a concentration of 20.1200 µg/L

min0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40

%

-15

85


CS05_0922_02 Smooth(Mn,8x2)centrifuge 10 min, 50µL 1M HCl

1.537e+004d3-IBA;1.00;826;10646;29

0.84

0.440.01

min

%

-15

85


CS05_0922_02 Smooth(Mn,8x2)centrifuge 10 min, 50µL 1M HCl

4.663e+0030.400.08 0.25 1.30IBA;1.00;68;1032;3 1.11

The Data Says It All- Successful Extraction From Plasma


Final extraction procedure - representative chromatogram of IBA at a concentration of 20.1200 µg/L

min1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

%

-15

85



1.099e+005d3-IBA;1.55;14997;107651;1112

1.25

min

%

-15

85



1.876e+005IBA;1.59;25435;182704;1303

1.26

The Data Says It All- Accuracy and Precision


LLOQ = 1.008 QC_L = 2.016 QC_M = 80.64 QC_H = 161.28

C [µg/L] d % C [µg/L] d % C [µg/L] d % C [µg/L] d %

0.8600 -14.68 2.1104 4.68 77.5662 -3.81 156.7754 -2.79

1.1218 11.29 2.1313 5.72 79.1035 -1.91 169.2208 4.92

1.1033 9.45 1.9815 -1.71 83.3323 3.34 153.4054 -4.88

1.1058 9.70 2.2560 11.90 82.9091 2.81 160.2228 -0.66

1.0152 0.72 2.1700 7.64 80.9280 0.36 168.3194 4.36

CV% 9.74 5.86 2.73 6.73

Table 1. IBA Between-Run Accuracy and Precision Data

The Data Says It All- Post-preparative Stability I


Nominal value QC_L = 2.0160 Nominal value QC_H = 161.2800

Back-calculated concentration= 2.0770 Back-calculated concentration = 157.3806

Name C [µg/L] d % Observed Change % Name C [µg/L] d % Observed

Change %

Val03_QC_L_PPS_04 1.9319 -4.17 -6.99 Val03_QC_H_PPS_04 172.1279 6.73 9.37

Val03_QC_L_PPS_05 1.7677 -12.32 -14.89 Val03_QC_H_PPS_05 159.4016 -1.16 1.28

Val03_QC_L_PPS_06 2.1579 7.04 3.90 Val03_QC_H_PPS_06 158.5488 -1.69 0.74

Average 1.9525 -3.15 -5.99 Average 163.3594 1.29 3.80

Table 2. Post-preparative pre-derivatization stability

The Data Says It All- Post-preparative Stability II



Back-calculated concentration= 2.0770 Back-calculated concentration = 157.3806


Change %

Val03_QC_L_PPS_01 2.0590 2.13 -0.87 Val03_QC_H_PPS_01 149.2222 -7.48 -5.18

Val03_QC_L_PPS_02 2.1517 6.73 3.60 Val03_QC_H_PPS_02 154.9090 -3.95 -1.57

Val03_QC_L_PPS_03 1.9520 -3.18 -6.02 Val03_QC_H_PPS_03 159.4893 -1.11 1.34

Average 2.0542 1.89 -1.10 Average 154.5402 -4.18 -1.80

Table 3. Post-preparative auto-injector stability

The Data Says It All- Long-term Stability



Back-calculated concentration = 2.0770 Back-calculated concentration = 157.3806


Change %

Val01_QC_L_LTS_01 2.1104 4.68 1.61 Val01_QC_H_LTS_01 156.7754 -2.79 -0.38

Val01_QC_L_LTS_02 1.9137 -5.07 -7.86 Val01_QC_H_LTS_02 159.7622 -0.94 1.51

Val01_QC_L_LTS_03 2.2070 9.47 6.26 Val01_QC_H_LTS_03 155.6041 -3.52 -1.13

Val06_QC_L_LTS_01 2.1700 7.64 4.48 Val06_QC_H_LTS_01 168.3194 4.36 6.95




Val07_QC_L_LTS_02 2.1960 8.93 5.73 Val07_QC_H_LTS_02 167.3102 3.74 6.31Average 2.1589 7.09 3.94 161.9544 0.42 2.91

Table 4. Long-term stability data

The Data Says It All- Calibration Curve Comparison


0

50

100

150

200

250

0 50 100 150 200 250

Calibration Curves from validation series

Val_01 Val_02 Val_03 Val_04 Val_06 Val_07

R2= 0,999692

R2=0,999019

R2=0,999057

R2=0,999255

R2=0,999279

R2=0,999263

The Data Says It All- Calibration Standards Data


Table 5. Representative Calibration Curves from the validation series

Calibration standardNominal conc. (µg/L)

CS011.0060

CS022.5150

CS035.0300

CS0410.0600

CS0520.1200

CS0650.3000

CS07100.6000

CS08201.2000

Val_01 1.0052 2.4971 5.1785 9.8920 19.9562 49.8166 100.2140 204.5654 Val_02 1.0195 2.5499 4.9616 9.9688 19.6790 49.5816 101.5344 212.8885 Val_03 1.0188 2.4558 5.0008 9.9627 19.623 49.7378 100.8191 213.2776 Val_04 1.0068 2.5380 5.0007 9.7848 19.8712 49.3275 103.3950 206.6564 Val_06 0.9980 2.5999 4.9028 9.8940 20.7158 49.6362 100.5640 205.9028 Val_07 1.0202 2.4060 5.1376 9.9172 20.0776 51.4172 100.7937 211.6671 Average 1.0114 2.5078 5.0315 9.9033 19.9871 49.9195 101.2200 209.1596Std 0.0093 0.0698 0.1057 0.0667 0.3952 0.7525 1.1502 3.8763d% 0.5 -0.3 0.0 -1.6 -0.7 -0.8 0.6 4.0


Conclusions

• Importance of pH adjustment during extraction

• Stability in matrix does not guarantee similar stabilitybehavior during extraction

• Successful liquid-liquid extraction results in morecost-friendly analysis


Professor Dr. Dobrin A. Svinarov, MD, Ph.D., Dr. Med. Sc.Head, Clinical Laboratory & Clinical PharmacologyChairman, Department of Clinical Laboratory & Clinical ImmunologyAlexander University Hospital, Faculty of Medicine Medical University of Sofia

Acknowledgements

Our bioanalytical team at MC “Comac Medical”Kichka KostovaMilena Tasheva

Desislava GeorgievaNedka SanyovaPavel Stoykov

overcomingextractionchallengesofabisphosphonate ...€¦ · bioanalytical department at comac...

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