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Automatic Multipumping Flow System for Handling Viscous Solutions:

Application to the Spectrophotometric Determination of Trimipramine

David S. M. Ribeiro â; João A. V. Prior â; João L. M. Santos â; José L. F. C. Lima â

a REQUIMTE, Serviço de Química-Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, Porto, Portugal

Online Publication Date: 01 January 2008

To cite this Article Ribeiro, David S. M., Prior, João A. V., Santos, João L. M. and Lima, José L. F. C.(2008)'Automatic Multipumping Flow System for Handling Viscous Solutions: Application to the Spectrophotometric Determination of Trimipramine',Analytical Letters,41:14,2684 — 2696

To link to this Article: DOI: 10.1080/00032710802363529 URL: http://dx.doi.org/10.1080/00032710802363529

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The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

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FLOW ANALYSIS

Automatic Multipumping Flow System for Handling Viscous Solutions: Application to the

Spectrophotometric Determination of Trimipramine

David S. M. Ribeiro, Joa˜o A. V. Prior, Joa˜o L. M. Santos, and Jose´ L. F. C. Lima

REQUIMTE, Servic¸o de Quı´mica-Fı´sica, Faculdade de Farmaacia, Universidade do Porto, Rua Anı´bal Cunha, Porto, Portugal

Abstract: The present work describes the implementation, by using MultiPump- ing Flow Analysis System (MPFS), of a spectrophotometric method for the determination of trimipramine in commercially available pharmaceutical formulations, based on the reaction with ammonium monovanadate in acidic medium yielding a colored compound with a maximum of absorbance at 620 nm. The improved flow mixing conditions during sample and reagents insertion and transport, as a result of the chaotic movement of the solutions originated by the MPFS pulsed flow, assured a fast reaction zone homogenization in a reduced residence time, which was particularly advantageous for carrying out analytical determinations that involved highly viscous solutions, as is the case of the sulfuric acid solution used in the determination of trimipramine, without impairing the sampling rate.

A linear working range for trimipramine concentrations of up to 50 mg L¹ (r¼0.9998;n¼6) was obtained, and the determined detection limit was about 1.15 mg L¹. The sampling rate was approximately 50 determinations per hour.

The obtained results were in agreement with those furnished by the reference procedure, with relative deviations lower than 4.7%. With the developed MPFS, the

Received 16 June 2008; accepted 10 July 2008.

Address correspondence to Joa˜o A. V. Prior, REQUIMTE, Servic¸o de Quı´mica-Fı´sica, Faculdade de Farmaacia, Universidade do Porto, Rua Anı´bal Cunha, 164, 4050–047 Porto, Portugal. E-mail: joaoavp@ff.up.pt

ISSN: 0003-2719 print=1532-236X online DOI: 10.1080/00032710802363529

2684

Downloaded By: [University of Porto] At: 16:30 18 November 2008

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consumption of the reagents ammonium monovanadate and sulfuric acid was reduced by approximately 41.02% and 54.29%, respectively, compared with a previously proposed multicommutated flow analysis system.

Keywords:Flow analysis, multipumping, pulsed flow, trimipramine, viscous solutions

INTRODUCTION

Trimipramine maleate, 5-(3-dimethylamino-2-methylpropyl)-10,11-dihy- dro-5H-dibenz [b,f] azepine acid maleate, is a tricyclic antidepressant agent that belongs to the dibenzoazepine class, with an anxiety-reducing sedative activity. Trimipramine is of particular value in depressed patients with insomnia and has been shown to be effective in the therapy of primary insomnia. Furthermore, as the pharmacological profile indi- cates, it might also be active as an antipsychotic (Gilman et al. 2001).

At present, the intensive therapeutical utilization of dibenzoazepine derivates increases the need to improve or develop new methods for their determination in body fluids and in pharmaceutical preparations, which, in this particular case, is further reinforced by the pharmaceutical relevance of trimipramine and its potential toxicity. Several methodologies for the chemical control of trimipramine in pharmaceutical formulations and biological samples have been proposed, involving liquid chromatography (Needham and Brown 2000), conductimetry (Nikolic et al. 1986), chemiluminescence (Wang et al. 2000, Greenway and Dolman 1999], spectrophotometry (Sane et al. 1983; Starczewska and Puzanowska-Tarasiewicz 1998; Hussein et al. 1989) and voltammetry (Ferancovaa et al. 2000; Ferancovaa et al. 2001).

In pharmaceutical analysis, the main aim is to develop fast, simple, versatile, and reliable methods that can be readily adapted for routine analysis at relatively low cost. In this context, flow analysis can assume a prominent role, and, in effect, a multicommutated flow procedure was already proposed for the spectrophotometric determination of trimipramine (Prior et al. 2003). However, these systems require the presence of separate equipment for propulsion of solutions, such as a peristaltic pump or a syringe pump, making the overall miniaturization of the manifold a difficult task and increasing its global cost and the complexity of the software controlling the automated system.

Recently, a novel flow analysis concept was proposed, based on the utilization of a pulsed flow, in opposition to the more typical laminar flow, which additionally allows a higher degree of simplicity in the com- ponents that comprised the flow system and, consequently, in the control

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of the system. Therefore, its application in the chemical control of pharmaceutical formulations, as an advantageous alternative to the reference methodologies, represents an attractive perspective.

The multipumping flow analysis methodology (Lapa et al. 2002) is based on the use of very small solenoid-actuated micropumps that are controlled by a computer, which makes possible a very simplified config- uration of the flow system, as well as its automatic control, because the basic operations in a chemical flow determination—namely, sample insertion, reagents addition, strategy for solutions mixing, and transport of the reaction zone toward the detector unit—are carried out by a single component, the solenoid micropumps being the only manifold active elements. The multipumping flow system (MPFS) performance is dependent on the characteristics of the pumping devices, which exhibit operational characteristics that support a good analytical performance:

e.g., they provide a nonmetallic, inert path for the dispensing of aggres- sive fluids, they are compact, accurate, and precise in the volumes that they dispense, and they are robust. The fully automatic control of these devices, under time-based and pulse-counting routines, makes MPFS an attractive methodology for implementation of reliable and versatile analytical alternatives for determination of pharmaceutical compounds, with the additional advantage of permitting a runtime access to important analytical parameters, such as flow rate, sample insertion, and reagent addition synchronization. Additionally, given that each micropump can be actuated individually in the fluids propulsion—each micropump being responsible for one solution—different sampling methods can be easily applied, such as merging zones, binary sampling, and single-sample volume. The characteristic pulsed flow originated by the actuation of the micropumps promotes a more efficient, reproducible, and improved sample=reagent intermixing than the one verified by exploring flow methodologies that rely on laminar flow, in which the interpenetration of solutions depends exclusively on the phenomena of diffusion and convection, making MPFS particularly advantageous, because it promotes an immediate reaction development and thus ensures an optimized analytical signal measurement.

This work evaluates, for the first time, the behavior and robustness of an MPFS when dealing with highly viscous and highly concentrated acid solutions. The reactivity between strong oxidants and the chemically active nitrogen atoms in the dibenzoazepine structure of the drug trimipramine, in acidic medium, yielding a blue-colored dimeric species (Mis- iuk 2000), was exploited using a fast, simple, and low-cost automated MPFS. This way, the main advantages of MPFS, such as ease of operation and versatility, high determination frequency, very low consumption of sample and reagents, and the improved hydrodynamic characteristics

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previously related, were applied for the spectrophotometric determination of trimipramine in pharmaceutical preparations.

MATERIALS AND METHODS Samples, Standards, and Reagents

All solutions were prepared with doubly deionized water, and analytical grade chemicals were used.

A 500 mg L¹ trimipramine stock solution was prepared by dissolving 69.72 mg of trimipramine maleate (Sigma-Aldrich¹) in 100 mL of a 4.0 mol L¹sulfuric acid solution. This stock solution was maintained under refrigeration. The working trimipramine standards (up to 50 mg L¹) were prepared daily by appropriately diluting the stock trimipramine solution with a solution of sulfuric acid 4.0 mol L¹.

A 510³mol L¹ ammonium monovanadate solution was prepared daily by dissolving 58.49 mg of NH₄VO₃(Merck¹) in 100 mL of a 4.0 mol L¹sulfuric acid solution.

The sample solutions, made with commercially available pharmaceutical formulations (Surmontil¹), were prepared by weighing and powder- ing a representative number of tablets. Thereafter, an appropriate amount of sample was dissolved in a solution of sulfuric acid 4.0 mol L¹

and filtered by gravity.

Apparatus

The designed flow manifold comprised three solenoid micropumps (Ref.

090SP Bio-Chem Valve Inc.), which were fixed displacement diaphragm pumps, dispensing a volume of 8mL per stroke. These micropumps (approximately 5 cm in length) are diaphragm pumps, operated by a solenoid, in which the diaphragm is maintained closed by means of an inner spring mechanism. When voltage is applied, the solenoid coil is activated in order to open the diaphragm. This opening action permits fluid to be drawn into the pump chamber. The fluid is dispensed from the pump by dropping the applied voltage, thus de-energizing the solenoid coil; the spring then forces the diaphragm back to the closed status.

Flow lines were made of 0.8 mm internal diameter (i.d.) polytetra- fluoroethylene (PTFE) tubing and used homemade end-fittings, connec- tors, and confluences.

The absorbance measurements were carried out in a LaboMed spec- trophotometer, model Spectro-22RS, at 620 nm equipped with a Hellma flow-cell, model QS 178.712 (18mL inner volume, 10 mm optical path).

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The analytical signals were registered through a Kipp and Zonen chart recorder, model BD111.

Automatic control of the analytical system was accomplished by means of a Pentium-based microcomputer and software, developed using Microsoft Quick-Basic 4.5. A lab-made electronic interface using a Cool- Drive^TM power drive board (NResearch Inc.) was used to activate the solenoid of micropumps through the LPT1 computer port.

Operation of the Flow System

The developed flow system exploiting the MPFS approach for the spectrophotometric determination of trimipramine is depicted in Fig. 1. The manifold employed three solenoid micropumps (P1,P2,P3), which were responsible for the individually handling of three different reagent solutions. The micropump P1 was responsible for insertion and propelling of the sulfuric acid solution, which was the carrier; P2 and P3 were used for inserting the ammonium monovanadate and sample solutions, respectively.

The analytical cycle started by actuating P1, inserting sulfuric acid solution in the analytical path in order to establish baseline, at a fixed pulse time of 0.2 s, corresponding to a pulse frequency of 300 min¹, which defined the flow rate as 2.40 mL min¹. Thereafter, micropump P1 was switched off, and, by actuation of micropumps P2and P3 at a fixed pulse time of 0.2 s, the ammonium monovanadate and sample solutions were simultaneously inserted with a preset number of micropump

Figure 1. Flow diagram for trimipaminc determination. C– Carrier solution (4.0 mol L¹H2SO4); R – reagent solution (5.010³mol L¹ammonium monovanadate in 4.0 mol L¹H₂SO₄); S – sample solution; P₁, P₂, and P₃– 8mL solenoid micro-pumps; x – confluence point; D – detector (620 nm); L – reactor coil.

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pulses [exploiting the flow merging zones approach (Bergamim et al.

1978)], which defined the sample volume inserted for the determination.

Thereafter, the micropumps P₂and P₃were switched off, and the estab- lished reaction zone was subsequently carried toward the detector by repetitive actuation of micropump P1and underwent an improved reaction development as a result of the fast intermixing between the small sample and ammonium monovanadate solution aliquots, originated by the micropumps actuation. In the flow manifold, the length of reactor was the minimum required to connect the confluence point X to the detector D. The analytical signal was monitored at 620 nm and recorded as a peak.

Reference Procedure

To assess the accuracy of the results of the developed procedure, pharmaceutical formulations containing trimipramine were analyzed following the reference procedure recommended by the British Pharmacopoeia (British Pharmacopoeia 2005). The Surmontil¹ tablets were weighed and powdered, and a determined amount of sample was dissolved in a mixture of equal volumes of acetonitrile and water and finally filtered.

The absorbance of the final solutions was determined by ultraviolet (UV) spectroscopy at 250 nm.

RESULTS AND DISCUSSION

The literature shows that dibenzoazepines have strong reactivity with several oxidizing agents because of the presence in their structure of chemically active nitrogen atoms (Misiuk 2000). Several oxidizing agents were proposed for the spectrophotometric determination of dibenzoazepines; however, some of them require extreme conditions in order for the reaction to take place, such as heating, agitation, and a long period for reaction development. The reagent ammonium monovanadate was revealed to be a good choice as oxidizing agent for the determination of dibenzoazepines, because it forms an intense blue- colored dimeric complex, with maximum wavelength absorbance at 620 nm. However, this reaction demands an extremely acid medium, obtained with a concentrated acid solution, which is cumbersome in its automation through flow methodologies because of the elevated acidity and viscosity of that acid solution. The use of solutions with elevated viscosity in flow methodologies is reflected in an inferior degree of mixing and, hence, in reaction development. Additionally, its

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transport through flow tubes implies a higher effort from the propulsion unit, demanding high robustness from that unit, which usually translates into a large, heavy propulsion unit, with elevated energy consumption. These characteristics represent an important drawback in automation and miniaturization of flow analysis systems.

With the goal of using, with full advantage, the potentialities for automation and miniaturization of the proposed methodology for the spectrophotometric determination of trimipramine, the chemical and physical optimization of the analytical system was carried out through a univariate approach, aiming for the maximization of the analytical signal.

Some preliminary assays revealed that the ammonium monovanadate and sulfuric acid concentrations of the solutions used in the determination markedly affect the amplitude of the analytical signal. The effect of sulfuric acid on the analytical signal was assessed over a concentration range from 2.0 to 6.0 mol L¹and the ammonium monovanadate concentrations were studied between 510⁴and 110²mol L¹.

The study of the influence of sulfuric acid concentration on the analytical signal was carried out using a sample volume of 80mL (10 pulses) of a 20 mg L¹trimipramine standard solution and a reagent volume of 80mL of a 110²mol L¹ ammonium monovanadate solution. The obtained results demonstrated that the analytical signal has an accentuated increase with the sulfuric acid concentration between 2.0 and 4.0 mol L¹, whereas, for higher concentrations, the signal tends toward stabilization. Therefore, for posterior assays, a solution of sulfuric acid was chosen, with a concentration of 4.0 mol L¹.

In the study of the influence of ammonium monovanadate concentration in the analytical signal, using the trimipramine solution of the previous assay and 10 pulses of the reagent solution in a 4.0 mol L¹ sulfuric acid solution, it was observed that the analytical signal markedly increased with reagent concentration from 1.010³to 5.010³mol L¹ and then tended toward stabilization. A 5.010³mol L¹ammonium monovanadate concentration was chosen for the following determinations.

The effect of reactor (L) length on analytical signal, placed between the confluence point X and the detector unit D (Fig. 1), aiming at extend- ing reaction development by increasing reaction time, was evaluated by inserting 10 pulses of a 20 mg L¹trimipramine solution and promoting different reaction conditions by carrying the reaction zone toward detection through flow reactors with different lengths: ‘‘0’’, 20, 30, 40, 50, 100, and 200 cm. The obtained results (Fig. 2) demonstrated that, by increasing the length of the reactor L, the analytical signal diminishes, indicating that dispersion prevails and that a higher signal was attained when using no reactor immediately before the detection unit, meaning that the mixture at the confluence point X was optimum. Despite the high viscosity of

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the sulfuric acid solution, these results confirm that the proposed MPFS, compared with the multicommutated flow system developed for the spectrophotometric determination of trimipramine, enables an improved, more efficient, and faster sample=reagent mixture with low radial dispersion because of a pulsed flow pattern caused by the micropumps actuation, allowing the accentuated reduction of the reaction coil length, at the same time, minimizing sample dispersion. Thus, the reactor L length used for the determination of trimipramine was ‘‘0’’ cm, corresponding to what was strictly necessary to provide the connection between the confluence point X and the detection unit.

Another relevant parameter that was optimized was the flow rate, because it determines the residence time of the reaction zone inside the flow system and hence the reaction time. The flow rate was evaluated by experimenting with different pulse times of 0.2, 0.3, 0.4, and 0.5 s, ori- ginating flow rates of about 2.40, 1.60, 1.20, and 0.96 mL min¹. It was verified that the analytical signal increased with the flow rate up to 2.40 mL min¹, confirming that an adequate reaction development was achieved prior to detection with a reduced resident time, thus enabling high determinations rates.

In MPFS, the insertion of predetermined sample volumes—by exploiting different sampling methods, such as merging zones, binary sampling, and single sample volume—is an operation that differs from other flow methodologies, because each micropump works individually in the insertion, commutation, and propulsion of the involved solutions, this procedure being easy to accomplish by modifying in the control software the actuation pattern and times of the micropumps. After assaying different sampling strategies (Fig. 3), it could be concluded that the

Figure 2. Influence of the reactor length on the obtained analytical signal.

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merging zones and binary sampling approaches improved the obtained analytical signal regarding the spectrophotometric determination of trimipramine. These results confirm that the merging zones and binary sampling strategies permitted a fast and efficient sample=reagent mixing, ensuring, at the same time, adequate reaction development.

At the same time, the last experiment related to the optimization of the sampling approach was used to evaluate the volume of sample solution to be inserted into the flow system, by assaying the influence in the analytical signal of the number of pulses of the micropump responsible for the sample solution insertion. After varying the number of pulses of the sample solution from 2 to 20 (corresponding to sample solution volumes between 16 and 160mL) with a pulse time of 0.2 s, the obtained results revealed a more pronounced increase in the analytical signal, up to approximately 10 pulses of sample solution, and, for a higher number of pulses, the corresponding increase in signal was less accentuated. In order to reach a compromise between determination rate and sensitivity, 10 sample pulses (80mL of sample solution) were selected for later determinations.

Interferences

In order to apply the developed methodology to the determination of trimipramine in pharmaceutical formulations, the influence of some compounds commonly used as excipients was assessed. A sample solution Figure 3. Influence of the number of sample pluses for distinct sampling strategies on the obtained analytical signal.&– Merging Zones;.– Binary Sampling;~– Unique Volumes.

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containing a fixed amount of trimipramine (20 mg L¹) and different concentrations of the excipients under evaluation were analyzed by the developed method. A compound was considered as noninterfering if the analytical signal variation was3% compared to the analytical signal obtained in the absence of the referred compound. The results revealed that the excipients (lactose, starch, talc, sodium croscarmellose, anhy- drous colloidal silica, sodium benzoate, and magnesium stearate) on a 100-fold molar ratio regarding trimipramine did not interfere.

Comparison of the proposed MPFS with MCFS

Some analytical figures of merit of the developed MPFS are reported in Table 1. Additionally, the proposed MPFS was also compared with another flow automatic system that exploited the multicommutation concept (MCFS). The MCFS developed for the determination of trimipramine in pharmaceutical formulations (Prior et al. 2003) was also based on the reaction of the drug with monovanadate in acidic medium.

According to Table 1, the obtained linear working range with MPFS was increased to up 50 mg L¹, and the sampling rate for the MPFS increased 92% over the determination rate of the MCFS.

The two flow systems were also compared in terms of consumption of reagents per determination. The MCFS required, per determination, 0.078 mg and 0.1373 g of ammonium monovanadate and sulfuric acid reagents, respectively, whereas the MPFS only required 0.046 mg and 0.0628 g of the same reagents. This means that the proposed MPFS allowed a significant reduction in the consumption of reagents, on the order of 41.02% and 54.29%, for ammonium monovanadate and sulfuric acid, respectively.

Table 1. Analytical figures of merit of the proposed multi-pumping flow system (MPFS) and the multicommutated flow system (MCFS) [11]

Analytical parameter MPFS MCFS [11]

Linear dynamic range (mg L¹)

Up to 50 1–18

Equation of linear calibration

A¼0.2886C0.3658 A¼0.7888Cþ0.6976 Detection limit

(mg L¹)

1.15

Sampling rate (h¹)

50 26

Not mentioned.

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Chemical Control of Pharmaceutical Formulations

The feasibility of the proposed system was ascertained by applying it to the spectrophotometric determination of trimipramine in commercially available pharmaceutical formulations with the following operating conditions: 10 sample pulses simultaneous merging with 10 pulses of 5.010³mol L¹ ammonium monovanadate solution, pulse time of 0.2 s (flow rate of approximately 2.40 mL min¹), 4.0 mol L¹ sulfuric acid carrier solution and no reactor coil. Using these parameters, a linear working response range for trimipramine concentration up to 50 mg L¹ was obtained.

The calibration curve was represented by A¼0.2886C–0.3658, where A was the peak height (expressed in cm) and C was trimipramine concentration (expressed in mg L¹), with a correlation coefficient of 0.9998 (n¼6). The detection limit was estimated as 1.15 mg L¹.

The obtained results (Table 2) were in agreement with those obtained by using the reference method, revealing a relative deviation less than 4.7%.

CONCLUSIONS

The analytical methods executed by exploring the MPFS approach are expeditious, easy to execute, and low cost, and they involve reduced production of residues. Additionally, the versatile operational characteristics of MPFS enables the analyst to perform different approaches and interventions in sample manipulation, such as sample conditioning, deli- vering, diluting, reagent addition, analyte separation or concentration—

assuring, at the same time, high reproducibility, without physical changes Table 2. Comparative results obtained in the determination of trimipramine in pharmaceutical formulation by the proposed and the reference method

Amount found (mg)

Sample Lot

Dosage mg=formulation

MPFS Methodology^a

Reference

method^a R.D. %^b

Surmontil A 25 25.00.2 25.50.8 2.0

100 103.80.6 104.01.5 0.2

B 25 24.30.2 25.50.5 4.7

100 99.90.8 102.31.6 2.3

aMeant0.05(Student’sttest)^SD^pﬃﬃ_n.

bRelative deviation of the developed method regarding the reference procedure.

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in the manifold. This automated flow methodology allowed an increase in the number of analyzed samples processed in time, reduction in expo- sure of the analyst to dangerous reagents, the elimination of errors associated with human manipulation of samples, an increment in analytical efficiency, the reduction in the consumption of reagents and production of residues, and the maximization of the analytical capacity of the labora- tory equipment. By exploring MPFS, all of these desirable characteristics encounter their full potential in real-life applications, where flow analysis manifolds should be as simple as possible.

In the developed work, the reaction with ammonium monovanadate for the spectrophotometric determination of trimipramine, implemented in MPFS resulted in a simple, low cost, sensitive, and precise methodology that could be used in routine analysis. Despite the use of a viscous solution of sulfuric acid, this did not impose any kind of hindrance to the determination of trimipramine in the MPFS, making these systems a good tool to implement reactions that generically involve, to some degree, viscous solutions.

The pulsed flow created by the actuation of solenoid micropumps, in combination with the merging zones or binary sampling strategies, pro- moted fast and very efficient sample=reagent mixing, reducing sample dispersion and ensuring a convenient reaction development, thus dispensing with the use of, for example, a mixture chamber.

As a result of the hydrodynamic characteristics of MPFS, the sample=reagent mixture achieved with the developed methodology in this work was superior to the one verified in the MCFS methodology previously proposed with predominantly laminar flow regime, thus enabling a high determination rate and flow rate without making use of a strategy of binary sampling associated with flow reversal during the sampling stage.

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