Development and Validation of a LC-MS² Method for the Simultaneous Quantification of Caffeine and Adenosine from DBS

 

Rajashri Lella, Elizabeth Mary Mathew, Sudheer Moorkoth*

Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal, Karnataka, 576104.

 

ABSTRACT:

A liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the simultaneous quantification of caffeine and adenosine in dried blood spot (DBS) samples. Caffeine is used in the treatment of apnea of prematurity in newborn children and adenosine levels play a major role in apnea. 40μL of whole blood spiked with the analytes was spotted onto specimen collection cards to prepare calibration DBS samples. 11.2 mm disks were punched from the card and was extracted in methanol and acetonitrile containing the internal standard. The extract was separated and quantified using a Phenomenex C18, 150x4.6mm, 3µm column as the stationary phase and 90 parts of 0.1% acetonitrile and 10 parts of 0.1% formic acid in water as the mobile phase delivered isocratically at 0.2mL/min. The injection volume and auto sampler temperature were 10µL and 35⁰C, respectively. The electrospray positive ion mode was used as the ionization source. The MS was operated using the single reaction monitoring at m/z 195→137.91 for caffeine and m/z 268→136.17 for adenosine. The method was seen to be linear from 0.050 -10µg/ml and demonstrated 85.12-88.98% recoveries for both the analytes. The method depicted an accuracy within 99.52-100.57% and the precision (coefficient of variation) was 0.32-1.66% for both the analytes.

 

 

INTRODUCTION:

Apnea of prematurity (AOP) is a common diagnosis in the neonatal critical care units. An apneic spell is defined as ‘the cessation of breathing for 20 seconds or longer or a shorter pause accompanied by bradycardia, cyanosis and palour’. Studies demonstrate a direct correlation of increased incidence of AOP with decreased gestational age. All infants born at ˂ 28 weeks were diagnosed with apnea in comparison to 20% of infants born after 30 weeks¹. AOP occurs as a result of physiological immature state of control during the fetal to neonatal transition. The underdeveloped central nervous system (CNS) in the immature neonate, results in a delay of spontaneous breathing. During this hypoxic episode the metabolic and neural activity within the brain leads to the formation of adenosine from adenosine triphosphate. Adenosine binds to its receptors and inhibit respiration leading to AOP².

 

Methyl xanthines are the main stay pharmacological treatment for AOP for over 40 years. Caffeine has gained wide popularity as the primary treatment of AOP owing to its efficacy, tolerability, wide therapeutic index and long half-life permitting once daily administration and safety margin³. Caffeine aids in the treatment of AOP by increasing the respiratory rate, contractility of the diaphragm, and by increasing the sensitivity to carbon dioxide. Caffeine is known to readily cross the blood brain barrier and enter the central CNS. Within the CNS, caffeine is known to exert antagonistic effects on the adenosine receptors A₁ and A_2A⁴_.

 

High performance liquid chromatography combined with ultra violet detector have been used for the quantification of caffeine^5,6 and adenosine^7–11 form different body fluids. It is known that mass spectrometry based techniques offer better sensitivity and selectivity for quantification of endogenous markers^12–17.

 

Dried Blood spot (DBS) is a micro sampling technique that is mainly adopted in newborns in the past few decades. DBS method is an ideal sampling technique for newborn and pediatrics considering its less invasive nature and minimal sample requirement. Additional advantages include convenience of storage and transport. Few studies have described the estimation of caffeine from DBS^12,18. There is no literature on the estimation of adenosine from DBS. Even though there is direct relationship between caffeine and adenosine in premature neonates, at present there is no literature to describe the simultaneous quantification of adenosine and caffeine from DBS. The aim of the present work was to develop a simultaneous LC-MS² method for the concurrent estimation of caffeine and adenosine from DBS.

 

MATERIALS AND METHODS:

Materials and Reagents:

Caffeine (99%) and Adenosine (˃99%) were procured from Sigma Aldrich (St. Louis, US). The internal standard Xanthine (minimum 99%) was procured from HiMedia Laboratories Pvt.Ltd (Mumbai, India). LC-MS grade acetonitrile was procured from Biosolve Chimie SARL (Dieuze, France). Formic acid 85% (AR grade) was procured from Merck (Kenilworth, US). In house Milli Q water (Siemens Ultra Clear) was used. Phenomenex C18, 150x4.6mm, 3µm was procured from Phenomenex (CA, USA).

 

Instrumentation:

A Thermo Scientific LC-MS (Massachusetts, USA) with Dionex Ultimate 3000 liquid chromatograph hyphenated with a linear ion trap analyser by an electron spray ionization source was used. MSⁿ and chromatographic method development were done using LTQ XL (Massachusetts, USA) and chromeleon (Massachusetts, USA) software respectively. Batch analysis was performed using the XCalibur software (Massachusetts, USA) and quantification using LC Quan (Massachusetts, USA).

 

Calibrators and quality controls:

We have adopted the methodology for the preparation of the calibration standards (CC’s) and quality control standards (QC’s) as per Vijay Kumar et al and Knapen et al^19,20. The calibrators and quality controls samples were prepared in left over blood from healthy controls after adjusting the haematocrit to 50% as that of a newborn. A 1000μg/mL primary stock solution of caffeine and adenosine was prepared in water. Suitable volumes of both the primary stock solutions were pipetted into a single volumetric flask to prepare a combined secondary stock solution (5μg/mL) in water. Subsequently, suitable dilutions of the primary and secondary stock solutions were performed to prepare calibrator stock solutions of concentration 1.25, 2.50, 5.00, 10.00, 20.00, 25.00, 50.00, 100.00, 200.00 and 250.00µg/mL in 10mL of water. Further, 40µL of the above calibration stocks were diluted to 1mL with blood to prepare calibration standards of concentration 0.05, 0.10, 0.20, 0.40, 0.80, 1.00, 2.00, 4.00, 8.00, 10.00µg/mL. The DBS calibration cards were prepared by pipetting 40 µL of the respective calibration standards onto 903 filter paper. Quality control DBS namely lower limit of quantification [LLOQ] (0.05µg/mL), lower quality control [LQC] (0.20 µg/mL), medium quality control [MQC] (1µg/mL), higher quality control [HQC] (8µg/mL) and upper limit of quantification [ULOQ] (10µg/mL) were also prepared in a similar manner. The Internal standard (IS) Xanthine was used at the concentration 1µg/mL for this study. After preparation, the DBS samples were dried at room temperature for three hours and shifted to envelopes with desiccants. The envelopes were then shifted to zip lock plastic bags and stored in -80°C until further analysis.

 

LC-MS method development and optimization:

Optimisation of MSⁿ conditions were performed by infusing separately both the analyte solutions (1µg/mL) at a flow rate of 10µL/min using the direct infusion pump in ESI (+) mode. During development, the mass scan filters were configured at a center mass of m/z 195, 268, 152.11 for caffeine, adenosine and xanthine (Internal standard) respectively with a width of m/z 10. For choosing the column, literature was studied and the C₁₈ column was chosen as the stationary phase. Phenomenex C18, 150x4.6mm, 3µm was seen suitable for the retention of both caffeine and adenosine within 10 min. Trials were initiated with a mobile phase consisting of 50 parts of 0.1% formic acid in water and 50 parts of 0.1% formic acid in acetonitrile at a flow rate of 0.2mL/min with ambient column compartment conditions. Adequate retention was observed for all the compounds, but we observed a peak shouldering for both the compounds. To optimize the chromatography we studied the increase in the organic component of the mobile phase from 50-90%. 90 parts of 0.1 % acetonitrile and 10 parts of 0.1% formic acid in water was seen to result in excellent peak symmetry (As=0.91). Column temperature (25- 40°C) was further investigated for its impact on run time. Increasing the temperature to 40°C was seen to favor elution within 10 min.

 

DBS extraction optimization:

For optimizing extraction, a 11.1mm disk was excised from the DBS and transferred to an eppendorf. The eppendorf was exposed separately to extraction with 50% v/v methanol, 100% v/v acetonitrile and 100% v/v acetone in water at different rotation speeds and temperature for 30 min. 50% v/v ACN and 50% acetone demonstrated a recovery less than 50% for all the analytes when compared to 100% v/v methanol. Further, to improve the recovery we performed trials using methanol in combination with varying parts (10-90% v/v) of acetonitrile. A recovery of 76.87-80.12% was observed for all the analytes in methanol: acetonitrile (80: 20% v/v). Further, to increase the recovery we altered the rotation speed (400-1000 RPM), rotation time (60-120min) and rotation temperature (25-45⁰C). Increase in rotation speed and rotation time was seen to improve the recovery for all the analytes. A maximum recovery of 85.12-88.98% was observed at 1000RPM within 90min. No increase in recovery was observed beyond 90min. Rotation temperature was found to negatively influence the percent recovery.

 

Method validation:

Method validation studies were investigated as per Food and Drug Administration guidelines²¹. All the CC’s and QC’s for the entire method validation study were prepared on a single day as per the Calibrators and Quality control section above. DBS samples were processed for investigating specificity by pipetting 40 µL of blood from six independent sources. Method parameters like limit of detection (LOD) and lower limit of quantification (LLOQ) were investigated based on the procedure of Gachet et al²². Blank subtraction technique was followed for the determination of linearity, recovery, accuracy and recovery. The linearity experiment was performed in five batches with each batch comprising of blank sample, zero sample and ten non-zero samples. The accuracy was investigated at LLOQ, LQC, MQC and HQC levels by comparing the mean calculated value (after endogenous subtraction) of six replicates at each QC to the nominal concentration at each level. Extraction recovery was assessed at LQC, MQC and HQC levels by comparing the responses (after subtraction of endogenous level) with neat standard solutions at each QC level. Matrix effect was investigated by the post extraction spike technique by comparing six replicates at LQC and HQC levels with corresponding neat standard solutions. We assessed carry over by injecting blank samples post ULOQ samples.

 

Influence of haematocrit on extraction recovery was investigated by six replicates of blood spots with adjusted haematocrits (30%, 35%, 40%, 45%, 50%, 55 %, 60% and 65%) at three quality control levels namely: LQC, MQC and HQC. A 11.1mm disc was transferred into an eppendorf and exposed to the optimized extraction and derivatization. The concentration of the analytes at different hematocrit values were investigated by the linear regression equation from a calibration curve produced by DBS standards at 50% hematocrit level.

 

Stability studies:

Stability studies were performed for all the analytes in stock and matrix as mandated by FDA²¹. Stock solution stability was investigated at MQC level by comparing freshly prepared neat stock solutions with stability samples at room temperature for 2, 4, 6 and 8 h. For stability studies in DBS, the QC’s were prepared at LQC and HQC levels and exposed to different stress conditions. The DBS samples were investigated for stability at room temperature (0, 0.5, 1, 1.5, 2, 4 and 7 h), for freeze thaw at 80°C (3 cycles), autosampler stability at 4°C (6, 12, 24 and 48 h ) and long term stability at -80°C ( 7, 15, 30, 60 days). At each time point we computed the mean and percent change.

 

RESULTS AND DISCUSSION:

Caffeine is a methylated xanthine alkaloid that occurs naturally. It acts as a CNS stimulant, myocardial stimulant and smooth muscle relaxant. Adenosine is a nucleotide formed by the glycosidic linkage of adenine and a ribose ring. The ability of linear ion trap mass spectrometry to perform selective reaction monitoring (SRM) enabled us to achieve advantage in terms of specificity. The MS² function in linear ion traps enabled separate SRM transitions for caffeine (195→138.1, CE:30), adenosine (m/z 268→136.17, CE: 35) and xanthine (152→94, CE:35). The protonated molecule of caffeine (m/z 195), and the internal standard Xanthine (m/z 152) fragments to produce product ions at m/z 95 by the loss of methyl isocynate (O––C––NCH₃) by a Retero Diels Alder agreement²³. Adenosine (m/z 268) forms adenine (m/z 136.1) by the loss of a protonated base and sugar moeity²⁴.

 

The optimized method contains a Phenomenex C18, 150x4.6mm, 3µm column preserved at a column temperature of 40⁰C. The mobile phase contains 0.1% formic acid in acetonitrile and 0.1% formic acid in water (90:10%v/v) at a flow rate of 0.2mL/min. The autosampler temperature and sample injection volume were 6⁰C and 10µL, respectively. The optimized mass spectrometry conditions were as following: spray voltage: 4.5kV, vaporizer temperature: 250°C, nitrogen sheath gas flow: 35 arbitrary units, auxiliary gas flow: 8 arbitrary units, sweep gas flow: 1 arbitrary units, ion transfer capillary temperature: 280°C with a voltage of 11.00 V and tube lens: 100V, multipole 00 offset:-5.65 V, lens 0: -8.40V, multipole 0 offset: -5.25V, lens voltage: -14V, gate lens: -76.00V, multipole1 offset: -11.20V, multipole RF amplitude: 400 V and front lent lens: -11.75 V. The retention time of caffeine, adenosine and xanthine (Internal standard) was 8.26, 7.20 and 5.67 min and the total run time of the method was 10 minutes. The optimized chromatogram is presented in Figure 1.

The optimised extraction method is as follows: A 11.2 mm disk was punched from the DBS and transferred to a centrifuge tube. 200µL of the internals standard solution prepared in methanol: ACN (80:20%v/v) was added to the Eppendorf tube. The sample was shaken for 90 min at 1000 RPM.

 

Figure 1: Optimized chromatogram representing the retention time (mins) for caffeine-8.26min, adenosine-7.20 min and methyl xanthine- (Internal standard) 5.67min.

 

The tube was then centrifuged at 10,000 RPM for 5 min at 4⁰C. 180 µL of the supernatant was separated and evaporated under nitrogen for 30min. The residue was reconstituted in 200µL of the mobile phase and 10µL was injected into the LC-MS² system.

 

The method was validated as per US FDA guideline²¹. The method was seen to be specific with the analytes and IS peaks completely resolved from the matrix under optimized chromatographic conditions. The method demonstrated a LLOQ of 50 ng/mL for both the analytes. We observed a %CV of 0.47 and 0.98 with % nominal of 101.49% and 103.26% for caffeine and adenosine respectively. The method was found to be linear from 0.050 - 10µg/ml with an r² of 0.9988 and 0.9987 for caffeine and adenosine. The Inter and intra-day precision % CV at various quality control levels was less than 1.66 and 1.25 % for caffeine and adenosine. Inter and intra-day accuracy was within 99.52-100.57% for both the analytes at different quality control levels. This method depicted recoveries of 80.61 - 86.54 % and 82.18 – 88.56 % for caffeine and adenosine respectively. The results of the different validation parameters were within acceptable limits ^{12,13,14,25,26,27}. The results are presented in Table 1.

 

Hematocrit in the neonatal period has a huge intra and inter individual variability. Hematocrit on the DBS is directly influenced by the viscosity of the blood. This is in turn known to influence the recovery of the analytes. No significant influence was found at the tested hematocrit levels (Table 2). Parameters like spot volume, punch size and chromatographic effect were not seen necessary to be performed as we have used an entire DBS sector (11.1mm).

 

Stability studies were performed by subjecting the analytes in stock and matrix to different stress conditions. For stock solution stability we detected a change of 2.77 and 1.67% for caffeine and adenosine respectively when stored in 2-8 °C for 30 days. This is within the specification limit of ±10 %. The bench top stability studies performed to understand the influence of laboratory conditions perceived a percent change of -1.19 % and -0.343 % for caffeine and adenosine respectively from the initial value. Further at HQC levels a percent change of -0.91 and -0.25 % from baseline was observed. The processed sample stability in the autosampler by 48 h depicted a percent change of -0.23 % and -0.67 % for caffeine and adenosine at LQC levels.

 

Table 1: Method performance specifications of caffeine and adenosine

LLOQ: lower limit of quantification, LQC: low quality control, MQC: medium quality control, HQC: high quality control

 

Table 2: Results of influence of hematocrit on caffeine and adenosine

Analyte	Percent recovery at different haematocrit
	30	35	40	45	50	55	60	65
Caffeine	85.12	83.28	84.12	83.78	88.23	83.23	85.12	86.12
Adenosine	81.23	82.45	83.23	84.24	83.23	83.45	83.09	84.67

 

Additionally, at HQC levels we detected a change of -2.31 % and -5.76 % for caffeine and adenosine respectively. The percent change after three freeze thaw cycles was less than -0.44 % at both LQC and HQC levels proving the freeze thaw stability. Long term stability study for a period of two months showed a percent change of only -0.12 % and -0.50 % for caffeine and adenosine respectively at LQC levels. At HQC levels these changes were -0.24 and -1.44% respectively. In all stability environments the percent change was within the specification levels of ±15 % signifying stability.

 

CONCLUSION:

A sensitive and specific analytical method based on LC-MS platform was developed and validated for the simultaneous analysis of caffeine and adenosine from DBS sample. The optimized extraction procedure showed good recovery of analytes from DBS. The selected hematocrit levels were not found to be affecting the levels of these markers. Results of stability studies were all within the acceptance limits. The data obtained in this study indicate the potential of the LC-MS² method to provide a selective method for the quantification of caffeine and adenosine concurrently from DBS samples of pediatrics and newborn affected with apnea.

 

ACKNOWLEDGEMENT:

The authors acknowledge Manipal College of Pharmaceutical Sciences (Manipal) and Department of pediatrics, Kasturba Medical College, Manipal for providing the infrastructure facility for carrying out this work.

 

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 23.05.2019           Modified on 21.06.2019

Accepted on 30.07.2019         © RJPT All right reserved