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Quantitative metabolite profiling of an amino group containing pharmaceutical in human plasma via pre-column derivatization and high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) Sanwang Li, Balázs Klencsár, Lieve Balcaen, Filip Cuyckens, Frederic Lynen, and Frank Vanhaecke Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04388 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 4, 2017

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Analytical Chemistry

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ACS Paragon Plus Environment


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Quantitative metabolite profiling of an amino group containing pharmaceutical in human plasma via pre-column derivatization and high-performance liquid chromatographyinductively coupled plasma-mass spectrometry (HPLC-ICP-MS) Sanwang Lia, #, Balázs Klencsár a, #, Lieve Balcaen a, Filip Cuyckens b, Frederic Lynen c, Frank Vanhaecke a a

Department of Analytical Chemistry, Ghent University, Campus Sterre, Krijgslaan 281-S12, 9000 Ghent,

Belgium b

Pharmacokinetics, Dynamics & Metabolism, Janssen R&D, Turnhoutseweg 30, 2340 Beerse, Belgium

c

Department of Organic and Macromolecular Chemistry, Ghent University, Campus Sterre, Krijgslaan 281-S4-

bis, 9000 Ghent, Belgium #

Both authors contributed equally to this work.

ABSTRACT Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Due to the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e. radiolabelling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on pre-column derivatization followed by HPLC-inductively coupled plasma-mass spectrometry (ICP-MS), was developed and validated. To introduce a suitable ‘hetero-element’ (defined here as an element that is detectable with ICP-MS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH2-groups. The presence of a known number of I atoms in both the drug molecule and its metabolites enabled a cross-validation of the newly developed derivatization procedure and quantification based on monitoring of the introduced Br. The formation of the derivatives was quantitative, providing a 4:1 stoichiometric Br/NH2 ratio. The derivatives were separated via reversed phase HPLC with gradient elution. Bromine was determined via ICP-MS at a mass-to-charge ratio of 79 using H2 as a reaction gas to ensure interference-free detection, and iodine was determined at mass-to-charge ratio of 127 for cross-validation purposes. The method developed shows a fit-for-purpose accuracy (recovery between 85% and 115%) and precision (repeatability 0.99 was found for both Br and I; additionally the 95% confidence interval of the intercept involves origo in both cases. The LoQ was defined as the concentration where a signal-to-noise ratio S/N ≈ 10 was obtained for the corresponding peak. The S/N ratio was determined based on USP . The LoQ values were 115, 90 and 90 µg Br / L in the form of T2, T3 and T4, respectively. It must be noted that the obtainable LoQ values should be significantly improved by using tetraiodophthalic anhydride (also commercially available) instead of tetrabromophthalic anhydride as derivatization reagent due to the approximately 40-fold higher ICP-MS sensitivity for I compared to Br (Table S-2). This approach was not followed here as a result of the presence of I in the drug molecule. Nevertheless, the LoQ values obtained are in good agreement with those reported for similar applications in the field. An LoD of 40 µg Br / L was found by de Vlieger et al63 in the analysis of Br-containing drug-related compounds using high-temperature liquid chromatography (HTLC) hyphenated to ICP-MS. LoQs of 40-65 µg Br / L and 120-180 µg Br / L were determined by Bendahl et al61 in the determination of Br-containing preservatives using UHPLC-ICP-MS and Meermann et al20 in the metabolite profiling of a novel Br-containing drug with HPLC-ICP-MS, respectively. An LoD of 90 µg Br / L was presented by Meermann et al20 in the same study based on radiodetection, thus demonstrating that the new approach (HPLC-ICP-MS) shows a similar sensitivity as the standard technique in the field (radiodetection). Accuracy and precision were evaluated by analyzing blank human plasma samples spiked with T2, T3 and T4 at four concentrations levels in a range of 0.1 to 0.7 mg/L of Br, corresponding to 0.1 to 1.1 mg/L of I in the final sample solution injected into the HPLC-ICP-MS unit (three replicate sample preparations at each level). The results of T2, T3 and T4 based on both Br and I are summarized in Table S-4 in the Supporting Information. As can be seen, apart from the LoQ level, the precision for each compound at each level was ≤13 RSD% based on Br and ≤12 RSD% based on I. Regarding the



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accuracy, the experimental result was typically within 90-110% of the reference value at all levels above LOQ for each compound based on either Br or I. From the results of the method validation, it can be concluded that an appropriately selective, linear, precise and accurate approach has been developed for the determination of levothyroxine and its metabolites in human plasma via the introduction of Br as a suitable element for ICP-MS detection into the molecules by means of precolumn derivatization followed by HPLC-ICP-MS analysis. Additionally the cross-validation performed based on I originally present in each target compound also confirmed the suitability of the newly developed approach.

CONCLUSION

To the best of the authors’ knowledge, this is the first demonstration of HPLC-ICP-MS-based quantitative metabolite profiling of a small-molecule drug in human plasma achieved via the introduction of a hetero-element into the target molecules by means of pre-column derivatization. The simple and straightforward type of chemical reaction, i.e. acylation of the free primary NH2-groups with a carboxylic anhydride used in this specific case potentially enables the development of a universally applicable approach for any primary amino group containing drug. This novel methodology could therefore prove to be a sterling alternative for radiolabelling. Although a successful application has been presented within the specific context of this study, this is just a starting point. In further studies for other drugs preferably also containing a secondary amino group, separation of the derivatized drug-related compounds from also derivatized endogenic amino group containing compounds will need to be accomplished. The nature of this chromatographic challenge can change case-by-case depending on the hydrophobicity (logP, logD) of the derivatized drug and its related compounds. In cases where compounds cannot be separated or are difficult to separate, 2D-LC separations can be considered as a possible tool to solve this issue. An additional, more general ICPMS-related problem is the continuously changing ICP-MS response when using gradient elution. In the present work, there was only approx. 12% difference in the acetonitrile content of the eluent in which the first and last peak of interest eluted, thus this effect could be neglected, as was confirmed by the successful accuracy assessment for each target compound. In case of a larger variation in polarity of the target compounds and thus, in eluent composition, this variation in sensitivity will need to be adequately addressed. Finally, also the development of derivatization approaches for other common functional groups i.e. carboxylic, alcoholic- and phenolic-hydroxyl groups typically present in pharmaceuticals is highly desirable 40.

AUTHOR INFORMATION

Corresponding Author *Email: [email protected]


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ACKNOWLEDGMENTS

Sanwang Li acknowledges the China Scholarship Council (CSC) for her PhD grant. Balázs Klencsár acknowledges the Special Research Fund of Ghent University (BOF-UGent) for his PhD grant.



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13 Fig. 1. Derivatization reaction of the free NH2-group of levothyroxine and its metabolites with tetrabromophthalic anhydride. levothyroxine (T4): R1 = I, R2 = I; 3,3’,5-triiodothyronine (T3): R1 = H, R2 = I; 3,5-diiodothyronine (T2): R1 = H, R2 = H I R1

R1

Br

O

I

O

Br

Br

NH2

HO

COOH

HO

+

O

O R2 O

Br

Br

I HOOC

Br

R2

Br

I NH

Br O

COOH

Millions

Figure 2. Investigation of the effect of the DMAP concentration in the reaction mixture: iodine chromatograms of derivatized standard mixture in the presence of 0; 0.3 and 1.5 mM DMAP Peaks: 1: T2; 2: T3; 3: T4; 4: primary derivative of T2; 5: primary derivative of T3; 6: primary derivative of T4, 7: secondary derivative of T2;8: secondary derivative of T3; 9: secondary derivative of T4

5 3

6

4

Intensity of 127I+ (CPS)

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2

7 1

9

2 3

8

1

0 0

5

10

15

Time (min) No DMAP

0.3mM DMAP

1.5mM DMAP


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14 Figure 3. Investigation of the effect of temperature: Iodine chromatograms of derivatized T4 standard at room temperature (black), 40 °C (blue) and 60 °C (red) Peaks: 1: Typical degradation products; 2: primary derivative of T4

x 100000

11

2

10 9

1


8 7

1

6 5 4

1

3

1

2 1 0 0

5

10

15

Time (min) R.T.

40°C

60°C

Figure 4. Investigation of the reagent concentration required for the conversion of T2, T3 and T4 105 100

Conversion (%)

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95 90

T2

85

T3 T4

80 75 70 0

5

10

15

20

25

Reagent concentration (mM)



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Figure 5. ESI-TOF-MS spectrum of the [M+H]+ molecule ion of the primary derivative of T4

+

I Br HO

O

Br

I HOOC

I

Br

+

I NH

Br O

COOH


H

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16

30 25 25 20

2

20

Thousands

x 100000

Figure 6. Selectivity in standard conditions. Iodine chromatogram of derivatized blank (blue), iodine chromatogram of derivatized T4 standard (black), bromine chromatogram of derivatized blank (green) and bromine chromatogram of derivatized T4 standard (red) Peaks: 1: T4; 2: primary derivative of T4

10 15 5 0 10

-5 -10

5

Intensity of 79Br+ (CPS)

15


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-15

1

-20 0

-25 0

5

10

15

Time (min) Iodine - T4 STD Bromine - T4 STD

Iodine - blank Bromine - blank



17

4

25

25

6

20

Thousands

30

5

15

20

10 5

15

0 -5

10

-10 5

Intensity of 79Br+ (CPS)

x 100000

Figure 7. Selectivity in human blood plasma. Iodine chromatogram of derivatized blank plasma (blue), iodine chromatogram of derivatized blank plasma spiked with T2, T3 and T4 (black), bromine chromatogram of derivatized blank plasma (green), bromine chromatogram of derivatized blank plasma spiked with T2, T3 and T4 (red) Peaks: 1: T2; 2: T3; 3: T4; 4: primary derivative of T2, 5: primary derivative of T3, 6: primary derivative of T4


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-15

1 2 3

-20 0

-25 0

5

10

15

Time (min) Iodine - spiked plasma Bromine - spiked plasma

Iodine - blank plasma Bromine - blank plasma


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