Untreated pain can have instant and prolonged consequences to behavioral and neurologic outcomes in neonates.1 The use of opioid analgesics is the most common analgesic strategy to minimize pain in neonates undergoing surgery.2,3 Nonetheless, side effects associated with opioid analgesics (e.g., respiratory depression, vomiting) often result in suboptimal treatment of postsurgical pain in this patient population.4 Regional anesthesia techniques may be an effective strategy to minimize postsurgical pain in neonates.5
The efficacy of the transversus abdominis plane (TAP) block to minimize pain has been demonstrated for specific surgical procedures in adults.6,7 However, the potential for the development of local anesthetic toxicity has brought the safety of TAP blocks into question.8 The use of the TAP block to minimize pain has been demonstrated in neonates,9 but no data regarding the safety of TAP block in neonates are currently available. Specifically, it is unknown whether plasma levels of local anesthetics are safe in neonates after TAP blocks. If proven to be safe, more clinical trials can be performed to evaluate the efficacy of the TAP block in this patient population.
The main objective of the current investigation was to evaluate blood bupivacaine concentrations in neonates having an ultrasound-guided TAP block. We hypothesized that TAP blocks would not result in unacceptably high local anesthetic concentrations in the blood when performed in neonates. We specifically sought to estimate 99% upper prediction limit of blood bupivacaine concentrations across multiple time points across 24 hours after the local anesthetic injection.
The study was a prospective, observational study. Reporting of the study was performed according to the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) consensus.10 Approval for the study was received by the IRB of the Ann and Robert H. Lurie Children’s Hospital of Chicago. Written informed consent was obtained from the parents or legal guardians of all participating subjects. Eligible subjects were neonates younger than 28 days undergoing any abdominal surgery. Subjects were excluded if they had significant cardiovascular disease, renal disease, liver disease, or if their weight was <1.65 kg.
General anesthesia was induced using propofol (1.5–2.5 mg/kg) and succinylcholine (1–2 mg/kg). An endotracheal tube was used to provide general anesthesia during the procedure. A TAP block was then performed by one of the investigators (SS) under ultrasound guidance using the posterior approach, as previously described, before the beginning of the surgery.11 The TAP block was performed with 0.125% bupivacaine with a total volume of 1 mL/kg. This dose is consistent with previous studies that demonstrated efficacy of the TAP block in the pediatric population.12 Whole blood samples were collected on a piece of Whatman Protein Saver 903 filter paper from either a peripheral line or a heel stick. Blood samples were obtained at 0, 5, 15, 30, 60, 120 minutes, 4, and 24 hours after the TAP block. When obtained from a heel stick, the samples were collected at the same time of glucose sampling to avoid unnecessary suffering for the patients. No additional needle sticks were performed for the sole purpose of the study.
The extracts from dried blood samples (DBS) collected on Whatman Protein Saver 903 filter paper were analyzed by iC42 Clinical Research and Development (Aurora, CO) using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system in combination with online extraction (LC/LC-MS/MS) based on a previously described assay platform for the quantification of drugs in DBS.13
After homogenization using a Bullet Blender (Next Advance, Averill Park, NY), 600 μL of the protein precipitation solution, methanol/0.2 M ZnSO4 (7:3, v/v) containing the internal standard (lidocaine) at a concentration of 25 ng/mL, was added to the samples. Samples were vortexed (2.5 minutes) and centrifuged (4°C, 16,000g, 10 minutes). After centrifugation, 200 μL of the sample supernatants was transferred into high-performance liquid chromatography (HPLC) vials and 200 μL of HPLC grade water was added.
Samples were analyzed using an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA)/AB Sciex API 5000 tandem mass spectrometry (LC-MS/MS, AB Sciex, Concord, ON, Canada) system including online extraction. The HPLC was interfaced with the API 5000 MS/MS by an electrospray ionization turboflow interface. The system was controlled, and data were processed using the Analyst 1.5.2 software (AB Sciex).
One hundred microliters of the extracted samples were injected onto a 4.6 × 12.5 mm online extraction column (Zorbax XDB-C8, 5 µm particle size; Agilent Technologies). Samples were washed with a mobile phase of 20% methanol supplemented with 0.1% formic acid and 80%, 0.1% formic acid. The flow was 5 mL/min. After 1 minute, the switching valve was activated and the analytes were eluted in the back flush mode from the extraction column onto a 150 × 4.6 mm analytical column filled with Zorbax XDB-C8 material of 5-µm particle size (Agilent Technologies). The mobile phase consisted of methanol supplemented with 0.1% formic acid and 0.1% aqueous formic acid. The following gradient was run: time 0 to 2 minutes: from 87% methanol to 100% methanol, 2 to 3.5 minutes: 100% methanol, 3.6 to 5 minutes: 87% methanol. The flow rate was 1 mL/min. The analytical column was kept at 65°C.
The mass spectrometer was operated in the positive ion mode. Mass spectrometry analysis was performed using the following parameters: collision gas = 12, curtain gas = 20, gas 1 = 50, gas 2 = 50, ion spray voltage = 5000, gas 2 heater = 500°C, entrance potential = 10, exit potential = 8.0. For bupivacaine, the declustering potential = 26, collision cell energy = 29, and for lidocaine, the internal standard, the declustering potential = 31, collision cell energy = 23. Nitrogen was used for electrospray ionization and zero air as collision gas. The analytes were detected in the multiple reaction mode and the following ion transitions were monitored: bupivacaine: m/z = 289.1 [M+H]+ → 140.2, lidocaine: m/z = 235.2 [M+H]+ → 86.2.
As considered fit for purposes of an exploratory clinical study, the prestudy assay validation followed the U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, and Center for Veterinary Medicine (2001). Guidance for the Industry. Bioanalytical Method Validation. Version May 2001.a In detail, the assay was validated following the fit-for-purpose principle and had the following key performance parameters as determined during prestudy validation: the range of reliable response ranged from 2.5 to 20,000 ng/mL, average interday accuracy was ±8.6%, and average interday imprecision was 6.6%. There were no significant matrix interferences, no matrix effects (ion suppression/ion enhancement), and no carryover. Extracted samples were stable in the autosampler at +4°C for at least 24 hours. Bupivacaine DBS were found to be stable at room temperature for at least 3 days and at +4°C for at least 1 week. Nevertheless, longer stability can be expected.14 To be on the safe side, DBS were stored at −70°C and below and were shipped on dry ice. During study sample analysis, the calibrators and their nominal concentrations correlated with r = 0.9975. Average accuracies of the quality control samples extracted and analyzed together with the study samples were 102.0% at 7.5 ng/mL, 107.0% at 37.5 ng/mL, and 90.3% at 375 ng/mL.
Normally distributed interval data are reported as mean and SD. Non-normally distributed interval and ordinal data are reported as median, range, or interquartile range. The Lilliefors test was used for the values from each subject at 30 minutes to confirm that the data followed a normal distribution (P = 0.47). The 99% upper prediction limit intervals for the plasma bupivacaine levels across subsequent times were calculated.
A convenience sample of 10 subjects was enrolled in the study. Patient characteristics are presented in Table 1. All neonates underwent uncomplicated ultrasound-guided TAP blocks. The time course of total plasma bupivacaine concentration is shown in Figure 1 (mean, 99% upper prediction limit interval). The highest 99% upper prediction limit interval occurred at the 30-minute interval, mean (99% upper prediction limit) of 0.13 µg/mL (0.38 µg/mL) in 4 of 10 subjects and the 99% upper prediction limit was below potentially toxic levels (1.5 µg/mL) across all times.15 For the remaining subjects, the highest blood levels occurred at other times (1 at 5 minutes, 1 at 15 minutes, 1 at 60 minutes, 2 at 120 minutes, and 1 at 240 minutes) but none crossed potentially toxic plasma levels. The highest individual concentration was 0.26 µg/mL and also occurred at the 30-minute interval. None of the neonates demonstrated any potential signs of local anesthetic toxicity.
The most important finding of the current investigation was the lack of toxic blood levels of bupivacaine when TAP blocks were performed to minimize acute pain in neonates undergoing surgical procedures. The 99% upper prediction limit for blood bupivacaine levels did not cross potentially toxic bupivacaine plasma levels across different times after the local anesthetic injection.8 Taken together, our results suggest a low risk of local anesthetic toxicity when TAP blocks are performed in neonates using a dose of 1.25 mg of bupivacaine per kilogram.
Our results are clinically important because postsurgical pain can have important implications to behavioral and neurologic outcomes in neonates undergoing surgery.2,3 Regional anesthesia may be an effective way to minimize postsurgical pain in neonates, but the potential development of adverse events (e.g., local anesthetic toxicity) may prevent the use of regional anesthesia in this patient population. To the best of our knowledge, this is the first study to prospectively evaluate local anesthetic plasma levels in neonates after a peripheral nerve block technique.
Major factors limiting pharmacokinetic studies in neonates are the sample volumes collected and the requirement for serial venous blood collections using a conventional approach, causing parents/legal guardians to frequently decline study participation. Nevertheless, minimally invasive, low blood volume collection strategies have emerged in recent years. DBS in combination with highly sensitive modern mass spectrometry technologies can be implemented using microvolume samples (≤50 µL) of capillary blood after a simple heel or finger stick and generating high precision to measure drug levels from DBS,16–18 including drugs relevant in the field of anesthesiology.19,20
Previous studies in the adult population have suggested that the TAP block can lead to plasma toxic levels of local anesthetics.8 Those studies use a greater dose of local anesthetics (2.5 mg of ropivacaine/kg) than that used by our group in the current study. Nonetheless, there is no evidence to suggest that larger doses of local anesthetics are more effective than the one used in the current study when a TAP block is performed in children.11
Our group has previously examined the safety of TAP blocks in children across several hospitals in the United States.21 We detected that 6.9% of children received potentially toxic local anesthetic doses, but none developed clinical signs of toxicity. Because the TAP block is performed under general anesthesia, early neurologic signs of local anesthetic toxicity are not easily identifiable. Although we demonstrated in the current study a low risk of local anesthetic toxicity when the TAP block is performed in neonates, other potential complications (e.g., visceral puncture, vascular injury) need to be further investigated.
Our study should be interpreted only within the context of its limitations. We did not examine the efficacy of the TAP block and, therefore, we cannot determine whether the analgesic efficacy of the TAP block results from plasma levels of local anesthetics as previously postulated by others.22 Nevertheless, the low levels of blood local anesthetics obtained in the current study support a local, rather than a systemic, analgesic effect. Future pharmacodynamic studies to evaluate the analgesic efficacy of the TAP block in neonates are warranted.
In summary, neonatal analgesic interventions remain an undeveloped field in perioperative medicine, and novel, minimally invasive, low-volume sampling techniques—in combination with LC-MS/MS—have the potential to remedy this situation. If proven to be safe, regional anesthetic techniques may be a promising strategy to minimize postsurgical pain in this patient population. Our results suggest a low risk of local anesthetic toxicity in neonates after a TAP block. The TAP block may be a safe strategy for minimizing neonatal postsurgical pain.
Name: Santhanam Suresh, MD.
Contribution: This author contributed to study design, conduct of the study, and manuscript preparation.
Attestation: Santhanam Suresh approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author.
Name: Gildasio S. De Oliveira, Jr., MD, MSCI.
Contribution: This author contributed to statistical analysis and manuscript preparation.
Attestation: Gildasio S. De Oliveira, Jr., approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.
This manuscript has been handled by: James A. DiNardo, MD.
The authors thank Jeffrey Galinkin, Christians Uwe, and Keith Hoffman from the Children’s Hospital Colorado for help with sample analysis and interpretation of the results and Angela Cambic for help in conducting the study.
a Available at: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf. Accessed January 3, 2015.
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© 2016 International Anesthesia Research Society
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